Establishing Native Plant Communities to Improve the Management of the Invasive Weed Mikania micrantha

Abstract

Using multiple species in native plant communities may improve control efficiency compared with single-species use. We conducted field investigations to assess the effects of Artemisia argyi, Portulaca oleracea, and their mixtures on the growth and reproduction of Mikania micrantha, followed by a greenhouse de Wit replacement series to compare different combinations of M. micrantha, A. argyi, and P. oleracea in terms of multispecies competition, phytoallelopathy, and photosynthesis. Field investigation showed that compared with M. micrantha monoculture (Group D), aboveground biomass, total stem length, flower biomass, inflorescence biomass, seed biomass, and seed number of M. micrantha increased in the P. oleracea community (Group B), though only seed number was significantly higher (p < 0.05). In contrast, in the A. argyi community (Group A) and the mixed community of A. argyi and P. oleracea (Group C), all these indicators decreased significantly (p < 0.05), in the order: Group C < Group A < Group D < Group B. This indicates that the mixed community (Group C) most strongly suppressed M. micrantha growth and reproduction. The effects of A. argyi, P. oleracea, and their mixture on the growth of M. micrantha in the greenhouse experiments mirrored the trends observed in field investigations. Calculated indices (relative yield, relative yield total, competitive balance index, and change in contribution) of A. argyi, P. oleracea, and their mixed population on M. micrantha demonstrated a higher competitive ability and higher influence of the combination of the two species compared with either A. argyi or P. oleracea alone. The interspecific phytoallelopathy experiment demonstrated strong allelopathic potential of A. argyi versus M. micrantha (p < 0.05) but showed no significant effect on P. oleracea. The net photosynthetic rate (Pn) of M. micrantha was generally lower in communities with both competitors compared with single-species communities. Our results suggest that, compared with a single plant population, the mixed population of A. argyi and P. oleracea exhibited a markedly enhanced ecological control capability through increased relative competitive ability, strengthened allelopathic inhibition, and markedly reduced photosynthetic efficiency of M. micrantha.

1. Introduction

Mikania micrantha, a perennial stoloniferous herbaceous vine, invaded China in the 1980s and is now recognized as one of the most serious invasive weeds in the world [1,2]. This weed is widely distributed in farmland, forests, orchards, roadsides, waste areas, and meadows throughout invaded regions in southern and southeast Asia and the Pacific Islands [3]. In invaded regions, the rapid growth, smothering, shading, and allelopathic effects of M. micrantha can lead to substantial economic losses in agricultural production and pose a significant threat to the biodiversity of these areas [3,4]. Over the past several decades, physical, chemical, and biological management approaches for M. micrantha have been attempted, achieving a certain degree of success [5,6,7]. Currently, chemical herbicides have been widely applied to control M. micrantha [8]. However, M. micrantha exhibits strong vegetative reproduction capacity [9] and establishes long-term soil seed banks in invasive area [10], enabling population regeneration even after herbicide spraying [11], which often leads to management failure. Furthermore, the cost of herbicide-based management is prohibitively high in some invaded regions [3,5]. Therefore, there is a global need to explore methods to control this invasive weed using ecologically sustainable approaches.

In natural ecosystems, plant communities with high species diversity typically exhibit greater resistance to invasion, whereas ecosystems with lower species diversity and simpler structures are more susceptible to invasion by alien species [12,13]. Using multiple species in native plant communities may improve control efficiency compared with single-species use. Exploring the underlying reasons and mechanisms is essential for developing effective strategies for invasive species management. Some plant species display greater growth rates and competitive advantages and can resist the establishment and spread of invasive alien weeds through competition for light, nutrients or allelopathic effects. For example, Pueraria lobata and Paederia scandens, climbing perennial vines native to China, were deliberately selected to suppress the recruitment of exotic plants in invaded habitats and were found to effectively control the invasive plant Ipomoea cairica over the long term, partly via reducing photosynthesis by I. cairica [14]. Many invasive weeds such as I. cairica and M. micrantha have high light requirements and are thus vulnerable to shading. Thus, exploring and harnessing the resilience of these native plant communities is crucial for effective ecological management and biodiversity conservation.

Over the past three decades, sciences have conducted systematic and extensive screening and assessment of potential native species, with the aim of achieving effective ecological management of invasive plants [15]. Ideally, such native species should not only exhibit robust growth and competitive advantages but possess high economic value and occupy similar niches as the targeted invasive alien species. For example, sweet potato (Ipomoea batatas), a high-value crop, has recently been used as an alternative to control invasive weeds through crop–weed competition [16]. While much research on competitive species has focused on the resistance of individual species to alien species, native plant communities may also exhibit strong resistance to invasive plants through the combined effects of diverse species within the community [17]. Therefore, studies on the resistance of native mixed populations to invasive weeds could be critical for developing innovative management strategies; however, there is currently a dearth of research in this area.

Based on the principle of limiting similarity, selecting native species that share the same habitats as the invader may be more effective [18,19]. Our previous field investigation found that Artemisia argyi and Portulaca oleracea often coexist with M. micrantha in the same habitats in the field and can be considered as belonging to the same functional group [20]. A. argyi, a perennial herb or subshrub native to China, exhibits strong competitive ability against other invasive weeds, such as Eupatorium adenophorum. One of the mechanisms identified making it an effective competitor is allelopathy, as A. argyi exhibits strong allelopathic effects on a number of weed species [21]. A. argyi is often utilized for the ecological restoration and replacement control of exotic invasive species [22]. P. oleracea, an annual herb native to China, is widely distributed in temperate and tropical regions and has strong ecological adaptability [23]. In China, P. oleracea is a common vegetable and is also often used as a Chinese medicine for the treatment of cardiovascular diseases [24]. However, knowledge of interspecific interactions among A. argyi, P. oleracea, and M. micrantha is limited.

Here, we conducted field investigations and greenhouse experiments to systematically evaluate the ecological control capabilities of single-species populations of A. argyi and P. oleracea, as well as their mixed population, against the invasive weed M. micrantha. Furthermore, this study integrated analyses of interspecific competition, allelopathic effects, and their influences on the photosynthetic characteristics of M. micrantha to elucidate the underlying mechanisms of action. Specifically, the objectives of this study were to further elucidate the resistance mechanisms of native plant communities towards the alien invasive plant M. micrantha and explore more sustainable management methods utilizing plant communities to suppress invasive plants.

2. Materials and Methods

2.1. Plant Material

Mikania micrantha, Artemisia argyi, and Portulaca oleracea are all present in Longchuan County, Dehong Prefecture, Yunnan Province, China. M. micrantha plants in our experiments were from a Longchuan population in Dehong Prefecture and maintained in the Agricultural Environment and Resource Research Institute greenhouse of the Yunnan Academy of Agricultural Sciences, Kunming, China. Seeds of A. argyi and P. oleracea were collected from local populations in September in 2022, dried for two months at room temperature, and then maintained stored at −4 °C. Although the three species, A. argyi, P. oleracea, and M. micrantha, have different geographic origins, conditions in Longchuan County, with average monthly rainfall of 1595 mm and an average monthly temperature of 18.9 °C, are highly favorable for the growth of all three species.

2.2. Site Characteristics, Field Surveys, and Data Collection

To clarify the characteristics of the growth and reproduction of M. micrantha in different species populations, we conducted a field investigation in the terrestrial ecosystem along the Nanwan River in Longchuan County (24°08–24°39′ N, 97°17–97°69′ E). This area is characterized by a typical tropical climate, having a rainy season featuring heavy rainfall with 90% humidity alternating with a dry season (see rainfall and temperature averages listed in Section 2.1). The vegetation surveyed was mainly herbaceous, without tall trees and shrubs. M. micrantha began to invade this region approximately 10 years ago [20]. In 2021, observation sites were established in the area, and systematic monitoring was implemented. In addition to M. micrantha, Artemisia annua, Crassocephalum crepidioides, Bidens pilosa, Portulaca oleracea, Commelina communis, Digitaria sanguinalis, Eleusine indica, and Borreria latifolia occur in this habitat.

According to the cover values of P. oleracea, A. argyi, and M. micrantha based on visual assessment, the surveyed communities were divided into four groups based on cover class. Group A indicated that the coverage of M. micrantha and A. argyi in the sample plot was approximately 40–50% each; Group B indicated that the coverage of M. micrantha and P. oleracea in the sample plot was approximately 40–50% each. Group C indicated that the coverages of M. micrantha, A. argyi, and P. oleracea in the sample plots were approximately 40–50%, 20–25%, and 20–25%, respectively; and Group D indicated that the coverage of M. micrantha in the sample plots exceeded 85%, while A. argyi and P. oleracea were absent.

We conducted field surveys from 1 to 5 December 2022. For each cover class, four 30 × 30 m field survey plots (replicates) were established. All plots were located in the same local area, with similar climate and altitude. Then, twenty-five 3 × 3 m quadrats were randomly selected in each plot for statistical analysis. Thus, a total of 400 quadrats were surveyed. For each quadrat, five plants of M. micrantha were selected randomly and carefully removed, separated, and dried (75 °C constant temperature for 72 h). Total shoot length, aboveground biomass, inflorescence biomass, and flower biomass were counted and measured. Here, we did not measure seed characteristics, because at this point in time, M. micrantha was still growing vigorously. On 16 January 2023, seed production of M. micrantha was measured in the study plots after flowering had waned, but prior to seed dispersal. Another twenty plants of each species were selected randomly and harvested within the middle region of each plot. The seed number and seed biomass of M. micrantha were measured.

2.3. Greenhouse Experiment and Data Collection

To test the effects of mixed populations constructed by A. argyi and P. oleracea on the growth and phenotype on M. micrantha, we conducted greenhouse experiments during the March–October growing season in 2023 at the National Modern Agricultural Experimental Base of Yunnan Academy of Agricultural Sciences, Kunming city (25°12–25°39′ N, 102°76–102°89′ E). On 20 March 2023, we sowed the seeds of three species in the nursery in the greenhouse. On 30 April, 180 seedlings of these species with similar height and size were selected and transplanted into 9 m² plots (3 m × 3 m) while maintaining a constant planting of 20 plants m⁻² (0.25 m × 0.20 m space) in each plot. Prior to the commencement of the experiment, the surface soil (0–12 cm) in each plot was thoroughly mixed and homogenized to ensure uniformity across all plots. The soil in the experimental area was red soil and weakly acidic, with a pH of 6.27. The nutrient composition of the soil was as follows: total nitrogen content of 1.12 ± 0.17 g·kg⁻¹, Olsen phosphorus content of 27.5 ± 0.93 mg·kg⁻¹, and available K content of 74.4 ± 3.16 mg·kg⁻¹.

To evaluate the resistance capacity of native populations to invasive alien species, we maintained the same number of native species and M. micrantha in each plot. In keeping with the principles of de Wit series competition experiments [25], we maintained a constant total density in the experiments, consisting of 180 plants grown in each 9 m² plot. To measure the effect of single-species native species populations, the ratio of A. argyi and P. oleracea to M. micrantha was 1:1 (90:90 plants were planted). For mixed native communities, the following three ratios of A. argyi, P. oleracea and M. micrantha were studied: 2:1:3/60:30:90 plants, 1:1:2/45:45:90 plants, 1:2:3/30:60:90 plants. We also set up trials with monoculture populations of A. argyi, P. oleracea, and M. micrantha at the same density. Thus, just as in the two-species de Wit series experiments, plant growth in monoculture for each of the three species could be compared with growth in mixtures via replacing part of the total density with either one other species or two other species to evaluate potential differences between the presence of single and multiple competitors with M. micrantha. All plants were distributed uniformly within the plot. All plots were arranged in a complete randomized block design with four replicates per ratio (total n = 32). A 1.5 m border was constructed between plots, and each plot was fenced with 0.35 m high glass panels to prevent the plants from climbing beyond the plots. Figure 1 shows greenhouse cultivation layouts for two-species and three-species interactions.

The experiment was terminated on 25 September 2023, 128 days post-transplanting. Twenty plants of each species were randomly selected and harvested from the central region of each plot. Main stem length, branch length, branch number, leafstalk length, leaf area, and aboveground biomass were recorded. Leaves were excised and analyzed using a leaf-area meter (Li-3000A; LI-COR Biosciences, Lincoln, NE, USA) to calculate the leaf area index. From June to September, net photosynthetic rate (Pn) measurements on leaves for M. micrantha were conducted midmonth using a Portable Photosynthesis System (LI-COR Biosciences LI6400XT, Lincoln, NE, USA) between 8:00 am and 11:30 am, with a 6400-02 LED source and 1000 μmol m⁻² s⁻¹ photosynthetically active radiation. During sampling, the CO₂ concentration in the surrounding air, air temperature, and relative humidity (RH) in the chamber were examined under natural conditions. Measurements were made on a representative leaf randomly chosen from five to six randomly selected individuals of M. micrantha in different treatments.

2.4. Interspecific Phytoallelopathy Effects

To evaluate the interspecific allelopathic effects of different species in mixed populations of A. argyi, P. oleracea, and M. micrantha, young leaves from the tops of A. argyi and P. oleracea plants were collected from the plant communities at 45 days after transplanting (15 June 2023). Leaf samples were cleaned with distilled water and cut into pieces (approximately 1–5 mm), then extracted according to a 5:1 mass ratio of distilled water to fresh leaves for 24 h. The extract was then filtered and evaporated in vacuo. After that, leaf water extracts were prepared and added to agar medium to create A. argyi and P. oleracea extract cultures of four concentrations, 0.0125, 0.025, 0.05, and 0.1 g/mL, at 50–55 °C. The control was agar medium containing distilled water only.

After that, the agar medium with different concentrations of leaf water extract was added to Amber wide-mouth glass bottles (150 mL). To ensure relative uniformity among the experimental stock, one-node segments (fresh weight 0.3–0.4 g, 5–6 cm pieces) were taken from central stem portions of relatively young A. argyi, P. oleracea, and M. micrantha plants of similar size and placed in the center of each bottle. Glass packer bottles were placed in an incubator with the following growth conditions: 20 °C, 12 h dark/25 °C, 12 h light. Experiments were carried out in 30 replicates for each concentration of each extract. Survival rate, shoot length, and aboveground biomass of each species were recorded after 15 days.

2.5. Statistical Analysis

Relative yield (RY) [26], relative yield total (RYT) [27], competitive balance index (CB) [28], and change in contribution (CC) [29] were calculated from final biomass (dry weight) for each species in each plot. These indices were calculated based on the final dry weight of each species in each plot using the following formulae.

Relative yield per plant of species a or b in a two-species culture with species b or a was calculated as follows: RY_a = Y_ab/Y_a; RY_b = Y_ba/Y_b.

Relative yield total for a two-species culture was calculated as follows: RYT = (RY_ab+ RY_ba)/2.

Competitive balance index for a two-species culture was calculated as follows: CBa = ln (RYa/RYb).

In the above equations, Y_ab(ba) is the yield for species a (or b) growing with species b (or a) (g/individual), Y_a(b) is the yield for species a growing in pure culture (g/individual), RY_ab(ba) measures the average performance of individuals in two-species cultures relative to pure cultures, and RYT is the weighted sum of relative yields for the two-species culture components.

Change in contribution for a three-species culture was calculated as follows:

$${\mathrm{CC}}_1^D={\displaystyle \frac{\raisebox{1ex}{$Y_{1(\mathrm{2,3}\dots n)}^D$}\!\left/ \!\raisebox{-1ex}{$\sum _{i=2}^n{Y}_{i1}^D$}\right.}{\raisebox{1ex}{${\left(p1\right)Y}_1^D$}\!\left/ \!\raisebox{-1ex}{${\sum }_{i=2}^n\left(pi\right)Y_i^D$}\right.}}-1$$

The calculation above is for species 1 grown in a mixture with species 2, 3 … n. The proportion at which species were grown is indicated by pi, where i is the species number. $Y_{1(23\dots n)}^D$ is the yield of species 1 (or species 2, or species n) grown at an overall density of D. ${CC}_1^D$ represents the proportion of biomass contributed by species 1 within a mixed population at an overall density of D.

We used the allelopathic index (IR) and synthetic allelopathic index (IR_SE) to evaluate the allelopathic interactions among M. micrantha, A. argyi, P. oleracea, and their mixed populations. These indices were calculated based on the shoot length and aboveground biomass in the target plant using the following formulae. Allelopathic index (IR) was calculated as follows: IR = (TR/CK − 1) × 100%, TR is the treatment and CK is the control (distilled water) [30].

Synthetic Allelopathic Index (IR_SE) was calculated as follows: IR_SE = (IR _shoot + IR _{aboveground biomass})/2, in which IR_SE > 0 or IR_SE < 0 indicates stimulation or inhibition of control [30].

Finally, we used the inhibition rate of aboveground biomass, survival rate, and specific leaf area to evaluate the impacts of M. micrantha, A. argyi, P. oleracea, and their mixed populations.

Inhibition rate of aboveground biomass (%) = ((A − B)/A) × 100%

Survival rate (%) = (Survival number of M. micrantha in treatment/Survival number of M. micrantha in control) × 100%

Specific leaf area (g·cm⁻¹) = Leaf area/Leaf biomass.

All growth variables (seed germination number, germination index, leaf area, spike number, seed number spike biomass, seed biomass and total biomass) and physiological variables (Pn, Gs, Ci, Tr, and WUE) of M. micrantha were analyzed by analysis of variance (one-way ANOVA) using the SPSS 23.0 software package (SPSS, Inc., Chicago, IL, USA). RY and RYT from each mixed culture were compared with the value of 1.00 using t-tests (p = 0.05 or p = 0.01), and values of RYT were tested for deviation from 1.0. Data were checked for homogeneity of variance. Treatment means were separated with Tukey’s HSD and post hoc multiple comparisons at the 0.05 significance level.

3. Results

3.1. Effects of Different Populations on Plant Growth and Productivity of M. micrantha in Field Survey

The field investigation revealed that M. micrantha in Group A and Group C habitats exhibited significantly lower aboveground biomass (19.14 g, 10.39 g), total stem length (596.75 cm, 513.56 cm), flower biomass (1.15 g, 0.58 g), inflorescence biomass (2.02 g, 1.07 g), seed biomass (0.31 g, 0.13 g), and seed number (4069.21, 2084.55) than those in the Group D habitat (p < 0.05). Notably, in the Group C habitat, all of the aforementioned metrics were at their lowest levels and showed significant differences compared with Group A and Group D (p < 0.05). However, in Group B, although M. micrantha exhibited higher aboveground biomass (28.29 g), total stem length (651.32 cm), and inflorescence biomass (4.60 g) compared with Group D, no statistically significant differences were detected between the two groups. Conversely, M. micrantha demonstrated significantly greater flower biomass (3.31 g) and seed number (13,481.63) in Group B than in Group D (

Table 1. The growth and reproductive traits of M. micrantha in various natural habitats.

Growth and Reproductive Parameters of M. micrantha	Biological Habitat Characteristics
	Group A	Group B	Group C	Group D
Aboveground biomass (g)	19.14 ± 2.11 b	28.29 ± 2.87 a	10.39 ± 1.58 c	27.26 ± 2.64 a
Total shoot length (cm)	596.75 ± 8.11 b	651.32 ± 10.87 a	513.56 ± 12.58 c	635.51 ± 7.64 a
Flower biomass (g)	1.15 ± 0.19 c	3.31 ± 0.78 a	0.58 ± 0.14 d	2.98 ± 0.57 b
Inflorescence biomass (g)	2.02 ± 0.12 b	4.60 ± 0.27 a	1.07 ± 0.15 c	4.43 ± 0.24 a
Seed biomass (g)	0.31 ± 0.08 b	1.01 ± 0.12 a	0.13 ± 0.05 c	0.93 ± 0.11 a
Seed number	4069.21 ± 395.93 c	13,481.63 ± 757.41 a	2084.55 ± 159.47 d	12,609.96 ± 684.96 b

Notes: Data are expressed as mean ± standard deviation; lowercase letters indicate significant differences (p < 0.05) within the same row. Identical letters mean no difference; different letters mean significant differences. Group A: M. micrantha and A. argyi each covered 40–50%; Group B: M. micrantha and P. oleracea each covered 40–50%; Group C: M. micrantha covered 40–50%, A. argyi and P. oleracea each covered 20–25%; Group D: M. micrantha exceeded 85%, with no A. argyi or P. oleracea.

).

3.2. The Effects of A. argyi, P. oleracea, and Their Combinations on the Growth of M. micrantha in the Greenhouse

As depicted in Figure 2, when M. micrantha was cocultivated with P. oleracea at a ratio of 1:1, its biomass (31.25 g), total shoot length (581.40 cm), and leaf area (20.54 cm²) were significantly higher than those of the monoculture population and other mixed cultivation populations. Specifically, there were significant differences in biomass and leaf area compared with the monoculture population and other mixed populations (p < 0.05). When M. micrantha, A. argyi, and P. oleracea were cocultivated at a ratio of 3:2:1, the biomass (12.29 g), total shoot length (458.33 cm), branch number (5.76), and leaf area (12.32 cm²) of M. micrantha reached their lowest levels, while its specific leaf area (882.72 cm²) reached its highest value (Figure 2A–E). Furthermore, specific leaf area exhibits significant differences compared with other populations (p < 0.05). Under monoculture conditions, the biomass of A. argyi and P. oleracea reached their maxima. No significant difference in biomass was detected between the monoculture population of A. argyi and its mixed cultivation counterparts (p > 0.05). In contrast, a significant difference in biomass was observed between the monoculture population of P. oleracea and its mixed-cultivation counterparts (p < 0.05), although no significant differences in biomass were found among the different mixed cultivation populations (p > 0.05) (Figure 2A–E).

3.3. Competitive Interactions

We assessed interspecific competition between M. micrantha and A. argyi, as well as between M. micrantha and P. oleracea, using the relative yield (RY), relative total yield (RYT), and competitive balance index (CB) (Figure 3A,B). When M. micrantha was grown in a 1:1 mixture with A. argyi, both the RY and RYT of the two species were significantly lower than 1.0, and the CB of M. micrantha was significantly below zero. In contrast, when grown with P. oleracea, the RY of M. micrantha was significantly higher than 1.0, while the RY of P. oleracea and the RYT of both species were significantly lower than 1.0, and the CB of M. micrantha was significantly above zero (Figure 3A,B). These findings indicate that A. argyi outcompeted M. micrantha where interspecific competition was stronger than intraspecific competition. Conversely, M. micrantha dominated P. oleracea where intraspecific competition was stronger than interspecific competition.

Furthermore, our study used the change in contribution (CC) to evaluate interspecific competition when all three plant species were grown together under different mixing ratios. Results showed that M. micrantha had consistently negative CC values (mix1: least negative; mix3: most negative) (Figure 3C). In contrast, A. argyi and P. oleracea showed positive CC values across all treatments, with significant differences among ratios (p < 0.05). A. argyi peaked in mix3, and P. oleracea, in mix1 (Figure 3C). These results show that the competitive contribution of M. micrantha was continuously suppressed (the suppression effect being strongest in mix1), while A. argyi and P. oleracea significantly enhanced their own contributions through interspecific competition, demonstrating a clear competitive advantage.

3.4. Interspecific Phytoallelopathy Effects

3.4.1. Allelopathic Effects of P. oleracea on M. micrantha and A. argyi

As shown in Figure 4, when the concentration of the aqueous extract of P. oleracea was below 0.05 g/mL, no statistically significant effects on survival rate, stem length and aboveground biomass of either M. micrantha or A. argyi. However, at a concentration of 0.1 g/mL, the extract significantly reduced both the survival rate and stem length of M. micrantha seedlings, with no significant effect on aboveground biomass (Figure 4A–C). At this same concentration, the survival rate of A. argyi was not significantly affected, whereas both stem length and aboveground biomass exhibited significant reductions (Figure 4D–F). The allelopathic comprehensive effect index analysis revealed that when the extract concentration exceeded 0.5 g/L, the allelopathic effect indices for both M. micrantha and A. argyi were negative, indicating a significant inhibitory effect. However, as the concentration decreased, these indices became positive, suggesting a slight promoting effect, but there was no significant difference (

Table 2. Interspecific allelopathic effects of aqueous extracts of A. argyi and P. oleracea on various species including M. micrantha in plant communities in greenhouse culture.

Synthetic Allelopathic Index	Concentration (g/mL)	Aqueous Extract of A. argyi		Aqueous Extract of P. oleracea
		M. micrantha	P. oleracea	M. micrantha	A. argyi
IRSE	0.1	−0.82 ± 0.11 d	0.12± 0.04 a	−0.13 ± 0.04 b	−0.12 ± 0.03 b
	0.05	−0.53 ± 0.06 c	0.06 ± 0.02 a	−0.03 ± 0.01 a	−0.07 ± 0.01 a
	0.025	−0.26 ± 0.07 b	0.06 ± 0.01 a	0.08 ± 0.02 a	0.07 ± 0.02 a
	0.0125	0.04 ± 0.01 a	0.03 ± 0.01 a	0.09 ± 0.01 a	0.03 ± 0.01 a

Different letters represent significant differences at p < 0.05.

).

3.4.2. Allelopathic Effects of A. argyi on M. micrantha and P. oleracea

As shown in Figure 5, when the concentration of the aqueous extract of A. argyi was 0.1 g/mL, the survival rate, stem length, and aboveground biomass of M. micrantha were significantly inhibited (Figure 5A–C). Specifically, the survival rate of M. micrantha decreased to 61.47% (Figure 5A), and the allelopathic index for M. micrantha at this concentration was −0.82 (Table 2). Under the same concentration, the survival rate of P. oleracea was significantly reduced, whereas both stem length and biomass exhibited significant increases (Figure 5D–F). The synthetic allelopathic index analysis revealed that, within the tested concentration range of the A. argyi aqueous extract, the allelopathic index of P. oleracea remained consistently positive (Table 2). In contrast, for M. micrantha, when the extract concentration exceeded 0.025 g/mL, the allelopathic index was consistently negative and decreased progressively with increasing concentration (Table 2). These findings indicate that A. argyi exerts a strong inhibitory allelopathic effect on M. micrantha, while promoting the growth of P. oleracea.

3.5. Photosynthesis Characteristics

From June to September, the photosynthetic rate (8.51, 10.89, 13.61, and 13.97 in June, July, August, and September, respectively) of M. micrantha under monocultural conditions was consistently higher than that of the population under mixed planting conditions (

Table 3. Net photosynthetic rate (Pn) of M. micrantha in different plant communities including A. argyi and P. olercea in June, July, August, and September 2020.

Species	Net Photosynthetic Rate (Pn) of M. micrantha
	June	July	August	September
Mm (Aa)	7.83 ± 0.19 b	8.57 ± 0.18 cb	11.22 ± 0.27 c	13.67 ± 0.11 a
Mm (Po)	8.43 ± 0.21 a	9.05 ± 0.23 b	12.96 ± 0.21 b	13.70 ± 0.13 a
Mm1 (Aa + Po)	7.05 ± 0.05 d	7.79 ± 0.08 d	8.68 ± 0.11 f	9.08 ± 0.49 c
Mm2 (Aa + Po)	7.26 ± 0.08 cd	7.97 ± 0.10 d	9.32 ± 0.25 e	10.29 ± 0.17 b
Mm3 (Aa + Po)	7.60 ± 0.16 bc	8.06 ± 0.06 d	10.09 ± 0.15 d	10.59 ± 0.29 b
Mm (mono)	8.51 ± 0.09 a	10.89 ± 0.17 a	13.61 ± 0.22 a	13.97 ± 0.15 a

Mm (Aa) signifies M. micrantha growing with A. argyi at 90:90 plants/m²; Mm (Po) signifies M. micrantha growing with P. oleracea at 90:90 plants/m²; Mm1 (Aa + Po), Mm2 (Aa + Po), and Mm3 (Aa + Po) signify M. micrantha growing with A. argyi and P. oleracea at 90:60:30, 90:45:45, and 90:30:60 plants/m², respectively; Mm (CK) signifies M. micrantha growing in monoculture. Different letters represent significant differences at p < 0.05.

). When the planting ratio of M. micrantha, A. argyi, and P. oleracea was 3:2:1, the net photosynthetic rate of M. micrantha was the lowest, significantly lower than that in monoculture at different stages. In addition, our study also found that the net photosynthetic rate of M. micrantha increased gradually from June to September under constant conditions (Table 3).

4. Discussion

The invasive plant M. micrantha frequently disrupts the ecosystem of invaded areas [2]. Using native species for ecological control is recognized as an environmentally sustainable approach [31]. However, most current research has focused on single-species control, while the potential of multispecies combinations remains limited. Numerous studies have demonstrated that diverse plant communities in natural ecosystems tend to resist alien plant invasions more effectively [32], indicating that multispecies combinations may offer a greater advantage than single-species approaches in controlling invasive plants. This study, through integrating field investigations and greenhouse experiments, demonstrated that mixed populations of A. argyi and P. oleracea significantly inhibited the growth and reproduction (p < 0.05) of M. micrantha more than single-species populations. Through the analysis of relative yield, relative yield total, competitive balance index, and change in contribution in A. argyi, P. oleracea, and their mixed population with M. micrantha, we found that although P. oleracea exhibited weaker interspecific competitive ability than with M. micrantha, its mixed population with A. argyi demonstrated higher competitive ability, with the combination of both species having greater influence than either A. argyi or P. oleracea alone. Furthermore, interspecific allelopathy experiments revealed that A. argyi exerted a significant allelopathic effect on M. micrantha (p < 0.05), with no notable inhibitory impact on P. oleracea. Additionally, this study showed that the net photosynthetic rate (Pn) of M. micrantha in the mixed population of A. argyi and P. oleracea was significantly reduced (p < 0.05) compared with its values in the single-species populations of either A. argyi or P. oleracea. Taken together, our study demonstrated that the combined application of A. argyi and P. oleracea exhibited enhanced suppression efficacy against M. micrantha compared with individual species, with underlying mechanisms elucidated through interspecific competition, allelopathic effects, and physiological trait analyses. Our research demonstrated that sustainable suppression of invasive alien plants, such as M. micrantha, can be achieved through strategic modulation of species combinations and community structure in affected habitats, providing novel ecological management frameworks for long-term efficacy.

Native species exert strong interspecific competition that plays a key role in resisting alien species invasion within ecosystems [33,34]. However, most current studies have focused on pairwise competitive interactions between single native and invasive species. The combined competitive effects of multiple native species on invaders are still poorly understood. In natural ecosystems, invasive species face complex competition from diverse native communities. The change in contribution (CC) quantifies shifts in the biomass contribution of individual species within mixed communities, thereby reflecting their relative competitive performance [29]. It is a useful tool for analyzing interspecific competition in systems with three or more species. A negative CC value indicates reduced contribution under competition, suggesting a disadvantage; a positive value indicates increased contribution, showing a competitive advantage. For example, Shen et al. used the CC index to assess competitive dynamics among sweet potato, hyacinth bean, their mixture, and M. micranthav [35]. The current study revealed that when A. argyi or P. oleracea were cocultivated with M. micrantha in equal proportions, A. argyi exhibited a stronger interspecific competitive ability than M. micrantha, whereas P. oleracea demonstrated a weaker competitive ability. In the mixed population comprising all three species, the change in contribution (CC value) of M. micrantha remained consistently below zero, indicating its persistent competitive disadvantage. In contrast, the change in contribution CC values of A. argyi and P. oleracea were consistently positive, reflecting their dominant competitive positions. Furthermore, analysis of M. micrantha’s growth and reproductive performance across different population types showed that it achieved the highest levels in the presence of P. oleracea alone and the lowest in the mixed culture of A. argyi and P. oleracea. These findings demonstrate that the cocultivation of A. argyi and P. oleracea significantly enhanced their competitive suppression of Mikania micrantha, effectively inhibiting its growth and reproduction.

Allelopathy plays a critical role in community assembly, which not only modulates competitive interactions among plant species but significantly influences the structural organization and functional dynamics of ecosystems [36]. Our study showed that A. argyi had strong allelopathic potential, but only M. micrantha was significantly inhibited, with no significant effect on P. oleracea. Thus, within a community comprising A. argyi and P. oleracea, M. micrantha was inhibited because of the allelopathic effect of A. argyi, while the growth of P. oleracea was relatively unaffected. This may allow P. oleracea to rapidly occupy available niche space, quickly diminishing resources available to M. micrantha. Our research indicates that by exploiting the differential allelopathic responses of M. micrantha and P. oleracea to A. argyi, innovative strategies can be formulated for constructing ecological communities with heightened resistance to M. micrantha invasion. Moreover, our study establishes a robust scientific foundation for facilitating ecological restoration in M. micrantha-invaded regions via community succession.

Strong ecological adaptability is one of the critical factors contributing to the successful invasion of M. micrantha [37]. This species can modify its leaf morphology to accommodate a wide range of environmental conditions. Plant functional traits are formed by plants adapting to different environments in the process of long-term evolution, balanced against constraints determined by overall plant structure and phylogeny [38]. Among the catalogue of plant traits, particular leaf traits of plants are closely tied to plant growth strategies and the ability of plants to acquire resources, reflecting survival strategies for adapting to environmental changes. Leaf area provides a key index to measure solar energy utilization efficiency of plants [39]. Greater specific leaf area may signify increased carbon assimilation capacity due to more leaf area produced for a given investment in biomass [40]. Our study showed that in all cultured plant communities including both P. oleracea and A. argyi, the leaf area and net photosynthetic rate of M. micrantha were significantly lower than when M. micrantha was grown with P. oleracea alone, A. argyi alone, and in monoculture. Although the leaf area index of M. micrantha index was diminished in the multispecies community, the specific leaf area was significantly higher than in other treatments. Higher rates of photosynthesis connected to higher leaf areas can lead to increased growth rates, biomass accumulation, and overall production. Higher carbon gain and growth may enable many invasive species to readily outcompete slower-growing species by facilitating colonization or resource acquisition. Thus, our study further revealed why the community comprising both A. argyi and P. oleracea had the greatest impact on M. micrantha among the combinations we tested from the perspective of plant leaf functional traits and photosynthetic characteristics.

5. Conclusions

Our results suggest that, compared with single-plant populations, the mixed population of A. argyi and P. oleracea exhibited a markedly enhanced ecological control capability through increased competitive ability, strengthened allelopathic inhibition, and markedly reduced photosynthetic efficiency of M. micrantha. Through field investigation and greenhouse experiments, it became clear that the plant community constructed of both A. argyi and P. oleracea had a greater competitive effect on M. micrantha than either species competing with M. micrantha on its own. The greatest inhibition of M. micrantha occurred when the population ratio among A. argyi, P. oleracea, and M. micrantha was 2:1:3. When M. micrantha was grown in a community with both A. argyi and P. oleracea, the photosynthetic capacity of M. micrantha was reduced and significantly impacted by allelochemicals produced by A. argyi, which had limited effects on P. oleracea. Thus, the niche of M. micrantha was restricted both by competition for resources and by allelopathy. Our findings suggest that fostering multispecies communities may provide a more potent and sustainable management strategy for suppressing M. micrantha than utilizing single competitors, especially if a variety of competitive mechanisms operate within the plant community. Potential multispecies systems should be tested utilizing modeling or experimental studies to determine how best to optimize the resistance of plant communities to invasion by M. micrantha. Once the best strategy for utilizing these native species is determined, efforts should be made to apply these findings in real-world agricultural settings to enable taking advantage of the combined competitive abilities of A. argyi and P. oleracea.