The Alien Invader, Rhus typhina L., Outperforms Its Native Competitor in the Scenario of Nitrogen Deposition and Arbuscular Mycorrhizal Fungi (AMF) Inoculation

Abstract

Nitrogen deposition has been proven to facilitate the establishment of alien plants. Previous studies have certified that nitrogen deposition enhances the resource availability of habitats and promotes the growth of alien invaders. Arbuscular mycorrhizal fungi (AMF) symbiose with vascular plants and assist plants in nutrient acquisition. AMF colonization has been proven to be another driving factor of plant invasion. However, few studies have integrated nitrogen deposition and AMF inoculation into the exploration on invasion mechanism. Based on a trait approach, the present study subjected the alien invader, Rhus typhina L., and its co-occurring native species, Acer truncatum Bunge, to nitrogen deposition and AMF inoculation and compared the phenotypic variation in aboveground and belowground traits in an inter-specific competition experiment. Through the effects of different nitrogen deposition and AMF infection on the functional traits of R. typhina and A. truncatum, the effects of mycorrhizal symbiosis between R. typhina and A. truncatum on abiotic factors and interspecific relationships were analyzed. We found that inter-specific competition stimulated the colonization of AMF in R. typhina, however, decreased the colonization rate of AMF in A. truncatum. Correspondingly, inter-specific competition significantly reduced the plant growth of A. truncatum as the aboveground morphological traits including plant height and crown area, and belowground traits including root length, root surface area, root volume, number of root tips, number of root tip branches and number of root cross decreased for A. truncatum. Nitrogen deposition promoted the shoot growth of R. typhina rather than that of A. truncatum. AMF inoculation significantly affected the belowground traits of A. truncatum as the root length and root surface area significantly decreased after AMF inoculation in the mixture planting of the two species. The chlorophyll content of A. truncatum decreased without AMF inoculation, while nitrogen deposition enhanced the net photosynthetic rate of R. typhina. The alien invader R. typhina outperforms its native competitor in the simulated scenario of resource fluctuation and facilitates its establishment. We speculate that AMF colonization promotes the extension of R. typhina rhizosphere and, thus, accelerates the growth and invasion of R. typhina.

1. Introduction

As a facet of Darwin’s naturalization conundrum, the limiting similarity hypothesis suggests that the invasion success of alien invaders is attributed to their difference from the native competitor in the introduced habitats [1]. Since the alien invaders take advantages in resource exploitation and stress tolerance, they can “defeat” the native neighbors and establish sustained populations. For instance, Leishman et al. [2] showed that invasive plants had higher leaf nitrogen and phosphorus concentration than native plants at the community and global level, which suggests a faster resource use for the invasive plants. van Kleunen et al. [3] presented that invasive plants had higher values in six trait categories of physiology, leaf-area allocation, shoot allocation, growth rate, size and fitness than native plants. Davidson et al. [4] found that invasive species almost always showed a greater phenotypic plasticity in their response to greater resource availability than non-invasive species. Liu et al. [5] demonstrated that invasive plants benefited more from global environmental change such as nitrogen deposition than the native plants. Mathakutha et al. [6] compiled the plant trait data from the sub-Antarctic Marion Island and found that invasive species differed from the native neighbors in key functional traits such as plant height and specific leaf area.

Anthropogenic activity boosts the nitrogen deposition to earth surface, and the deposition rate has been expected to increased one to two folds by the middle of 21st century [7]. Nitrogen deposition usually enhances the resource availability of recipient habitats [8]. Corresponding to the fluctuation resources hypothesis on invasion mechanism, the positive effects of nitrogen deposition on plant invasion, which probably threatens the stability of local ecosystem and causes challenges to the sustainable socioeconomic development, has been certified by simulated experiments from the mesocosm to field [9]. For instance, Valliere et al. [10] showed that nitrogen enrichment exacerbated the loss of native plants and indirectly increased the cover and biomass of nonnative annuals. Wang et al. [11] reported that sediment eutrophication induced a higher trait plasticity of leaf nitrogen concentration and chlorophyll concentration for the invasive Alternanthera philoxeroides, which enabled the invasive species to outcompete the native congener A. sessilis. Luo et al. [12] found that phenotypic plasticity of traits such as leaf area and belowground biomass explained the competitive advantage of the invasive population of Plantago virginica over its native population. Dangremond et al. [13] presented that nitrogen enrichment accelerated the invasion of mangroves into saltmarshes by favoring the growth and reproduction of mangrove rather than those of saltmarsh plants. Obviously, plant traits can predict the invasive outcome for alien invaders, and trait comparison between alien invaders and their neighboring natives can decipher the invasion mechanism [14]. Successful invaders usually equip with fast growth and situate at the fast side of the trait economic spectrum [15]. Therefore, plant species with a higher growth rate (e.g., a larger body size), a greater photosynthetic efficiency (e.g., higher values in net photosynthetic rate and chlorophyll content) and a larger root network (e.g., greater root length, root surface area, root volume, root tips and root crosses) commonly show competitive advantages in space exploitation and resource utilization [16,17,18]. Environmental stimulus such as nitrogen deposition further promotes the competitive performance of alien invaders such as faster growth and greater resource use.

The contribution in plant nutrient assimilation, water uptake, pathogen defense and stress tolerance of arbuscular mycorrhizal fungi (AMF) has been broadly realized [19,20,21]. Considering the mutualistic relationship between plants and AMF, AMF can also gain carbon from the root exudation of plants [22]. Despite the above benefits, the effects of AMF on plant competition especially between the exotic and native species are uncertain. Several studies have shown that exotic plants “attract” more AMF colonization and facilitate their performance [23,24,25]. Thus, exotic plants take advantage from AMF symbiosis over native plants [24,25]. However, the inoculation of arbuscular mycorrhizal fungi potentially assisted the native species Bidens biternata in resisting the invasion of Bidens alba [26]. A meta-analysis has reported that invasive plants have a probability to be poorly colonized by AMF and gain less benefit from AMF [27]. Therefore, the competitive outcome between alien invader and its co-existing native counterpart is uncertain when facing AMF colonization.

Rhus typhina L. is an exotic woody species with great invasive potential and has been introduced into the landscape architecture and forest plantation in North China [28]. It has the characteristics of fast growth rate and strong adaptability to harsh environments. Since this species can release allelopathic chemicals to the environment, the competitive advantage of this species over the native neighbors may be augmented [29]. Previous studies have revealed the trait advantage of R. typhina in changing environments of nutrient resource and water. For instance, Yuan et al. [30] found that R. typhina outcompeted the native competitor, Quercus acutissima Carr. in carbon gain and nutrient assimilation under different soil N:P ratios. Guo et al. [31] showed that the native Cotinus coggygria suffered more from drought stress than R. typhina, since the photosynthetic apparatus of C. coggygria appeared to be more severely harmed by drought. Wang et al. [32] indicated that R. typhina had a higher phenotypic plasticity than its native congener under heterogeneous nitrogen deposition condition, and, thus, an advantage in environmental acclimation may exist for R. typhina. However, few studies have considered the probable interactive effects of nitrogen deposition and AMF inoculation on the trait performance of R. typhina, especially nitrogen deposition may restrict the positive effects of AMF colonization on nutrient assimilation and pathogenic defense [33,34]. Acer truncatum is a deciduous tree native to China and has high ornamental value. A. truncatum and R. typhina are usually jointly used in plantations and coexist in the field. The present study compared the phenotypic variation between the alien invader, R. typhina, and its native competitor, Acer truncatum Bunge, under simulated condition of nitrogen deposition and AMF inoculation. We hypothesized that R. typhina takes advantage of faster growth and more efficient resource use compared to A. truncatum.

2. Materials and Methods

2.1. Experimental Procedure

The seeds of two woody species (Rhus typhina L. and Acer truncatum Bunge) were collected from Mount Lao (36°19′ N, 120°62′ E) in November, 2019, and the seeds were stored at 0–4 °C during the winter. The following treatment was performed to stimulate the germination of the two species in February, 2020. The seeds of R. typhina were washed with 70 °C baking soda solution for 10 min and then washed with tap water. The seeds of R. typhina and A. truncatum were soaked in tap water for 24 h. In the artificial climate chamber (temperature 23–25 °C and humidity 60–70%), the soaked seeds of each species were then placed on trays with sterilized (121 °C, 30 min) turf soil for germination in 7–14 days. During this period, we sprayed water into the tray every day to keep all the seeds moist and reduce the water loss. Healthy seedlings for each species were selected and transplanted into plug plates. When most seedlings of each species in the plug plates had three or four fully-developed leaves, seedlings of the two species with similar size were transplanted into plastic pots (23 cm in diameter and 23 cm in height) for further treatments.

Each pot was filled with a mixture of 1:1 (v/v) of sandy loam and peat (specification: 10–30 mm, pindstrup substrate, Denmark). And the mixture of substrate had been autoclaved (121 °C, 2 h). The total weight of the substrate was 5.0 kg. Debris and stones were removed carefully and completely.

The AMF (Funneliformis mosseae) was provided by the Mycorrhizal Biotechnology Institute of Qingdao Agricultural University. Trifolum repens was propagated as a host plant for 4 months, and its fungal spores, mycelium and mycorrhizal root segments were used as AMF inoculants.

2.2. Experiment Set-Up

The mesocosm experiment was set up in the greenhouse at Chengyang Campus, Qingdao Agricultural University (36°00′ N, 120°03′ E) on 10 March 2020. The average temperatures inside the greenhouse during plant growth were 22–25 °C, and the average relative humidity was 65–75% in a 11:13 h light: dark photo-period. Qingdao is located in the temperate region of China with a hybrid climate type of monsoon climate of medium latitudes and marine climate. The average temperature is 12.7 °C, and the average annual precipitation is 700 ± 100 mm [32].

Three treatments including AMF inoculation, planting pattern and nitrogen deposition were set for the experiment. Autoclaving was employed to kill all soil biota before treatments. The treatment of AMF inoculation includes (1) no AMF are inoculated (0 AMF) and (2) AMF are inoculated (+AMF). For inoculation treatments, 8000 units of inoculum potential (IP) of F. mosseae were distributed to pots according to the experimental design by mixing in with the soil. The units of IP were measured with the methods described by Liu and Luo [35] and calculated as IP = N × W × K + S, where IP is the inoculation potential, N Is the number of vesicles in the root segment per unit length, W is the root weight (g), K is the root length per unit mass (cm) and S is the number of spores in the inocula per unit mass or volume. The uninoculated controls received 50 g of autoclaved inocula in each pot. The treatment of planting pattern includes (1) the mono-planting of R. typhina, (2) the mix-planting of the two species and (3) the mono-planting of A. truncatum. This treatment included one plant in each pot for the mono-planting of R. typhina and the mono-planting of A. truncatum, and one of both plants in the pot for the mix-planting of the two species. The treatment of nitrogen deposition was designed as: (1) no nitrogen deposition (CK, N1), (2) mild nitrogen deposition (8 g Nm⁻²·yr⁻¹, N2) and (3) severe nitrogen deposition (20 g Nm⁻²·yr⁻¹, N3) [36,37]. The present form of nitrogen deposition in China is inorganic, and the ratio of ammonia nitrogen to nitrate nitrogen is 2:1. (NH₄)₂SO₄ and KNO₃ were used in the present study to simulate the nitrogen deposition with a ratio of 2:1. Since sulfur and potassium are, respectively, constituent elements of (NH₄)₂SO₄ and KNO₃, KCl and K₂SO₄ were used to equalize the content of sulfur and potassium in different treatments of nitrogen deposition. The nitrogen was added once every Monday at 5 PM, and 100 mL nitrogen solution was added in each pot each time. The prescription and concentrations of added solutions followed that shown in

Table 1. Concentration and composition of three nitrogen treatment solutions.

Nitrogen Treatment (g m−2 Year−1)	Solution Composition	Concentration (mol L−1)
0	K2SO4	3.80 × 10−4
8	(NH4)2SO4	1.52 × 10−4
	KNO3	1.52 × 10−4
	K2SO4	2.28 × 10−4
	KCl	1.52 × 10−4
20	(NH4)2SO4	3.80 × 10−4
	KNO3	3.80 × 10−4
	KCl	3.80 × 10−4

. Each treatment was replicated for 10 times in our experiment.

2.3. Harvest and Measurement

On 20 September 2020, the experiment ended and the aboveground traits, AMF colonization rate and belowground traits were measured.

2.3.1. Aboveground Traits

Two aboveground morphological traits, including plant height and crown area, were immediately measured and calculated when the experiment ended. The plant height was measured as the vertical distance of the plant base to the apical bud. The crown area was calculated as follows:

crown area = 0.5 × a × b

a represents the maximal horizontal length of the plant crown, and b represents the width of the plant crown perpendicular to a.

On a sunny and cloudless day, the net photosynthetic rate (A) was measured using the Li-6800 photosynthesis-fluorescence measurement system (LI-COR, Inc., Lincoln, NB, USA). The third and fourth leaves from the tip of each replicate were selected. The air flow through the leaf chamber was set to 400 μmol·s⁻¹, the chamber temperature to 27 °C and the CO₂ concentration to 400 μmol·mol⁻¹. A was measured at a photosynthetic photon flux density (PPFD) of 1000 μmol·photons m⁻² s⁻¹ provided by a blue-red LED light source mounted above the leaf cuvette.

One leaf on the third leaf node from the tip of each individual plant was selected for the determination of chlorophyll content using the ethanol method. Approximately 0.1 g of fresh leaf tissue was ground in 10 mL of 95% ethanol with calcium carbonate and quartz sand until all the leaf tissue dissolved. The absorbances of the supernatant were measured at 649 nm (A₆₄₉) and 665 nm (A₆₆₅) using a spectrophotometer after 24 h of dark treatment. Chlorophyll a content (Chl_a), chlorophyll b content (Chl_b) and chlorophyll content were calculated separately as (Wang et al., 2019) [38]:

Chl_a = (13.95 × A₆₆₅ − 6.88 × A₆₄₉) × 0.01/leaf fresh weight

Chl_b = (24.96 × A₆₄₉ − 7.32 × A₆₆₅) × 0.01/leaf fresh weight

chlorophyll content = Chl_a + Chl_b

2.3.2. AMF Colonization Rate

The AMF colonization rate was determined following the method by Liu and Chen [39]. The roots of each plant replicate were sliced into 0.5–1 cm long fragments, and then the fragments were placed into a test tube. A KOH solution with a 5–10% concentration was added into the test tube, and then the test tube was water bathed for 20–60 min at 90 °C. After water bath, the roots were soaked in the HCL solution with a 2% concentration for 5 min. The roots were stained using acid fuchsin stain with 0.01% concentration after the acid was removed from the roots. Then, the stained roots were water bathed for 20–60 min at 90 °C. Color separation was performed on the roots with lactic acid. Microscopic examination was used to observed the AMF colonization rate. The AMF colonization rate was calculated as follows:

$$\mathrm{AMF} \mathrm{colonization} \mathrm{rate} (\%)=\frac{0\times {\mathrm{n}}_0+10\%\times {\mathrm{n}}_1+20\%\times {\mathrm{n}}_2+\dots +100\%\times {\mathrm{n}}_{10}}{{\mathrm{n}}_0+{\mathrm{n}}_1+{\mathrm{n}}_2+\dots +{\mathrm{n}}_{10}}$$

n₀ represents the root fragment number without AMF colonization, n₁ represents the root fragment number with an AMF colonization rate of 10%, n₂ represents the root fragment number with an AMF colonization rate of 20%, n₁₀ represents the root fragment number with an AMF colonization rate of 100%.

2.3.3. Belowground Traits

The roots were harvested carefully using slow water flush to prevent the mass loss. Then, the roots were rinsed using deionized water to remove small rocks and other substances. Three lateral roots each replicate were selected and scanned. Then, the root length, root surface area, root volume, number of root tips, number of root tip branches and number of root cross were analyzed using WinRhizo Pro 2009 (Regent Instruments Inc., Québec City, QC, Canada).

2.3.4. Statistical Analysis

All trait data met the assumption of normality and homogeneity of variance prior to analysis. A two-way ANOVA was performed to test the effects of planting pattern and nitrogen deposition on the AMF colonization rate. A three-way ANOVA was applied to test the effects of the planting pattern, nitrogen deposition and AMF inoculation on aboveground traits and belowground traits of the two species each. If a significant treatment effect was detected, post hoc pair-wise comparisons of means were performed to examine the differences between treatments using Duncan’s test for multiple comparisons. All data analyses were conducted using SPSS 25.0 (IBM Corp., Armonk, NY, USA).

3. Results

Planting pattern and nitrogen deposition had significant effects on the AMF colonization rate of R. typhina independently and interactively, while the significant effect on that of A. truncatum was only detected in planting pattern (

Table 2. F values and significances of two-way ANOVA of planting pattern and nitrogen deposition on the arbuscular mycorrhizal fungi (AMF) colonization rate of Rhus typhina and Acer truncatum.

	Planting Pattern (P)	Nitrogen Deposition (N)	P × N
R. typhina	981.118 ***	21.063 ***	32.751 ***
A. truncatum	42.203 ***	0.904 ns	2.635 ns

*** p < 0.001, ^ns: no significant difference.

).

Considering aboveground traits, AMF inoculation had significant effects on the net photosynthetic rate of both species and on the crown area of A. truncatum (

Table 3. F values and significances of three-way ANOVA of arbuscular mycorrhizal fungi (AMF) inoculation, planting pattern and nitrogen deposition on the aboveground traits of Rhus typhina and Acer truncatum.

	AMF Inoculation (A)	Planting Pattern (P)	Nitrogen Deposition (N)	A × P	A × N	P × N	A × P × N
R. typhina
Height	0.160 ns	7.154 *	27.113 ***	0.043 ns	0.305 ns	0.729 ns	0.208 ns
Crown area	0.073 ns	17.930 ***	4.956 *	0.039 ns	0.095 ns	0.119 ns	0.547 ns
A	346.458 ***	6.731 *	944.958 ***	53.538 ***	2.356 ns	19.181 ***	0.226 ns
Chlorophyll content	2.289 ns	1.290 ns	68.462 ***	3.591 ns	12.732 ***	4.852 *	3.461 *
A. truncatum
Height	3.207 ns	473.578 ***	25.163 ***	0.447 ns	4.956 *	2.413 ns	9.761 ***
Crown area	4.516 *	740.433 ***	18.173 ***	1.331 ns	2.294 ns	0.292 ns	13.151 ***
A	10.649 **	3.474 ns	20.586 ***	1.829 ns	4.661 *	0.874 ns	2.218 ns
Chlorophyll content	0.022 ns	19.243 ***	40.275 ***	4.887 *	0.038 ns	0.491 ns	0.061 ns

A—net photosynthetic rate. * p < 0.05, ** p < 0.01, *** p < 0.001, ^ns: no significant difference.

). The planting pattern had significant effects on the height and crown area of both species, while respective significant effects of planting pattern on net photosynthetic rate and chlorophyll content were found for R. typhina and A. truncatum (Table 3). Nitrogen deposition had significant effects on all aboveground traits including height, crown area, net photosynthetic rate and chlorophyll content of both species (Table 3). The interactive effects of each two treatments and the three treatments on the aboveground traits were mostly insignificant (Table 3).

Considering belowground traits, AMF inoculation had significant effects on all traits of both species except root tip number (

Table 4. F values and significances of three-way ANOVA of arbuscular mycorrhizal fungi (AMF) inoculation, planting pattern and nitrogen deposition on the belowground traits of Rhus typhina and Acer truncatum.

	AMF Inoculation (A)	Planting Pattern (P)	Nitrogen Deposition (N)	A × P	A × N	P × N	A × P × N
R. typhina
Root length	133.528 ***	8.256 **	21.747 ***	48.508 ***	3.072 ns	15.767 ***	10.735 ***
Root surface area	41.524 ***	12.565 **	6.776 **	4.208 *	2.572 ns	0.466 ns	4.340 *
Root volume	22.502 ***	29.057 ***	63.420 ***	3.857 ns	0.202 ns	1.319 ns	1.429 ns
Root tip number	2.246 ns	64.494 ***	34.906 ***	1.172 ns	8.582 **	1.592 ns	0.014 ns
Number of root tip branches	51.012 ***	1.686 ns	3.010 ns	3.675 ns	1.201 ns	4.319 *	1.059 ns
Root cross number	7.669 **	3.217 ns	68.090 ***	14.696 ***	4.589 *	3.379 *	19.564 ***
A. truncatum
Root length	70.921 ***	3386.634 ***	17.924 ***	328.094 ***	0.276 ns	28.437 ***	0.234 ns
Root surface area	5.850 *	932.307 ***	13.149 ***	165.633 ***	1.703 ns	11.582 ***	1.072 ns
Root volume	21.700 ***	538.551 **	1.034 ns	64.479 ***	0.024 ns	0.119 ns	0.336 ns
Root tip number	2.275 ns	79.349 ***	124.938 ***	21.218 ***	7.105 **	45.599 ***	16.902 ***
Number of root tip branches	26.933 ***	2108.211 ***	68.770 ***	265.475 ***	0.260 ns	44.744 ***	1.492 ns
Root cross number	223.437 ***	12,835.811 ***	80.174 ***	998.432 ***	13.230 ***	14.100 ***	35.184 ***

* p < 0.05, ** p < 0.01, *** p < 0.001, ^ns: no significant difference.

). The planting pattern had significant effects on all traits of R. typhina except the number of root tip branches and root cross number, while the effects of the planting pattern on the belowground traits of A. truncatum were totally significant (Table 4). Nitrogen deposition exerted significant effects on all traits of R. typhina except number of root tip branches, while the significant effects of nitrogen deposition were found on all traits of A. truncatum except root volume (Table 4). The significant interactive effects of treatments on belowground traits appeared to be found more for A. truncatum than for R. typhina (Table 4). For instance, AMF inoculation and planting pattern had significant interactive effects on all belowground traits of A. truncatum; however, the above two treatments had significant interactive effects on merely three belowground traits including root length, root surface area and root cross number (Table 4).

3.1. Inter-Specific Competition

The AMF colonization rate was significantly higher under mix-planting than under mono-planting for R. typhina. However, the contrast was shown for A. truncatum (Figure 1a,b). Under different nitrogen deposition levels, the effects of AMF on R. typhina were different. The infection rate of mix-planting R. typhina at N2 was the highest, and the infection rate of mono-planting R. typhina at N2 and N3 was higher than that of N1. The mycorrhizal infection rate of A. truncatum was not affected by the nitrogen deposition.

No significant difference in plant height and crown area was shown for R. typhina between under the two planting conditions, while significant decreases in plant height and crown area were shown for A. truncatum under mix-planting condition (Figure 2a–d). The chlorophyll content of A. truncatum decreased without AMF inoculation under inter-specific condition, while that of R. typhina showed an inconsistent response to competition under different conditions of nitrogen deposition and AMF inoculation (Figure 2g,h).

Belowground traits, including root length, root surface area, root volume, number of root tips, number of root tip branches and number of root cross, decreased for A. truncatum under mix-planting condition, while R. typhina showed a more complex response of belowground traits to inter-specific competition (Figure 3a–l).

3.2. Nitrogen Deposition

The plant height generally increased, while the crown area was consistent with nitrogen deposition for R. typhina (Figure 2a–d). A. truncatum displayed an inconsistent response in plant height and crown area to nitrogen deposition under separate conditions of inter-specific competition and AMF inoculation (Figure 2a–d). For instance, a decrease in plant height was shown in the treatment of mono-planting without AMF inoculation; simultaneously, decreases in crown area were shown in the treatments of mono-planting with AMF inoculation and mix-planting without AMF inoculation (Figure 2a–d). The net photosynthetic rate of R. typhina increased significantly with the nitrogen deposition in all treatments of planting pattern and AMF inoculation, while that of A. truncatum was consistent when AMF were not inoculated (Figure 2e,f).

3.3. AMF Inoculation

No significant variation in plant height and crown area was shown for R. typhina under different treatments of AMF inoculation (Figure 2a,c). The plant height of A. truncatum significantly decreased under N2 and mono-planting treatment with AMF inoculation, while a significant increase in plant height was shown under N3 and mono-planting treatment with AMF inoculation (Figure 2b). The crown area of A. truncatum significantly increased under N1 and mono-planting treatment with AMF inoculation, while a significant decrease in crown area was shown under N3 and mono-planting treatment with AMF inoculation (Figure 2d).

Both root length and root surface area significantly increased with AMF inoculation for the two species under mono-planting conditions (Figure 3a–d). However, R. typhina showed either an increase or a maintenance in root length and root surface area with AMF inoculation under mix-planting conditions, while a significant decrease in root length and root surface area was shown for A. truncatum under mix-planting conditions (Figure 3a–d).

4. Discussion

4.1. R. typhina “Lured” a Greater AMF Colonization and Showed Advantage in Body Size When Facing Competition

The mycorrhizal infection rates of R. typhina and A. truncatum were different with different nitrogen levels, which might be species-specific. Prior studies have proposed that the alien plant invaders can enhance their invasive potential by enhancing mutualists, for instance symbiosing with mycorrhizal fungi, and, thus, a positive plant–soil feedback was shown between the alien plant invaders and mycorrhizal fungi [40,41]. The symbiosis causes a win-win consequence for both the alien plant invaders and the mycorrhizal fungi, since the plant invaders can provide carbon sources for mycorrhizal fungi and the mycorrhizal fungi actually extend the root network of the alien plant invaders and show assistance in nutrient assimilation, water uptake, pathogen defense and stress tolerance [19,20,21,22]. The specific studies on R. typhina have also found that the colonization of this alien invader altered the soil fungal communities and facilitated the soil fungi [42,43]. The present study showed that the inter-specific competition enhanced the colonization rate of AMF for the alien invader, R. typhina. Since AMF can provide assistance such as nutrient assimilation and stress tolerance to plants, AMF can be considered as a category of environmental resource. Therefore, R. typhina “lured” a greater AMF colonization and intercepted more environmental resources when competing with A. truncatum. From this perspective, the alien invader, R. typhina apparently outcompeted the native counterpart, A. truncatum, in exploiting AMF, which agrees with the above studies.

Body size has been commonly considered to determine the competitiveness for space and resource of organisms, especially in the facets of space occupation and resource use and thus affect the invasion of alien plant invaders [18]. Obviously, the plants with greater height and larger canopy take advantage in space occupation both vertically and horizontally. Simultaneously, such plants likely outcompete with their counterparts with smaller size in resource use of solar radiation, since such plants are prone to intercept more light resource [18], which is partly and indirectly supported by the observation on chlorophyll content, as inter-specific competition decreased the chlorophyll content of A. truncatum without AMF inoculation. The present study showed that the alien invader R. typhina maintained the plant height and crown area when facing competition, while the native A. truncatum showed decreases in these traits correlated with body size. Subsequently, the chlorophyll content of A. truncatum decreased when facing competition with R. typhina without AMF inoculation. Obviously, the larger body size conferred an advantage of light resource use on R. typhina. Simultaneously, maybe the larger crown of R. typhina shades the A. truncatum in the treatment of mixing planting, which causes the decrease of light availability for A. truncatum. Hence, the competitiveness for space and resources was downgraded for the native species. However, the alien invader displayed a stability in the competitiveness for space and resource. A prior study by Yuan et al. [30] reported a similar result in a competition experiment by R. typhina and another native counterpart Q. acutissima, as inter-specific competition promoted larger plant height and crown area for R. typhina. However, it exerted insignificant effects on those traits of Q. acutissima. Hence, the alien invader performed better in aboveground morphology than the native counterpart, which is probably a mechanism for the establishment of R. typhina.

Considering the root traits, inter-specific competition negatively affected the growth and development of the native species, as all measured root traits including root length, root surface area, root volume, number of root tips, number of root tip branches and number of root cross decreased under mixed plating condition, while the root trait response of the alien invader was inconsistent. The belowground competition from R. typhina significantly encroached the space for the belowground exploitation of A. truncatum and caused a more negative effect on the root growth and development of native A. truncatum. The maldevelopment of roots directly restricts the capacity of nutrient assimilation [16,17]. Therefore, the native plant, A. truncatum, likely captured less nutrients when facing competition with the alien invader, R. typhina. As a consequence, the capacity of nutrient utilization declined more prominently for the native species, which may indirectly lead to the competitive advantage of the alien invader in soil nutrient utilization.

4.2. Nitrogen Deposition Did Not Shrink the Body Size of R. typhina Compared to A. truncatum

Nitrogen deposition in a certain range can enhance the photosynthetic rate and accelerate the plant growth since nitrogen, as a limiting nutrient element on earth, is correlated with the synthesis of photosynthetic apparatus and enzymatic activity [44]. However, the overload of nitrogen can reduce net photosynthetic rate and may decrease the growth rate via decreasing the chlorophyll concentration and Rubisco activity in leaves [45,46]. The present study found that the body size of the alien invader did not shrink following the nitrogen deposition, since an increase in plant height and a consistency in crown area were present, which partly corresponds to the increasing net photosynthetic rate. However, the native A. truncatum displayed a decrease in body size under several conditions. Nitrogen deposition acted as a promoter for the performance of R. typhina, and R. typhina can be considered as a resource-demanding species. However, excessive nitrogen input restricted the shoot growth of native species and created stressful environments for A. truncatum, which indicates the strong adaptability of A. truncatum to harsh habitats. Since the nitrogen deposition appears to be endless in the future scenario [47], the expansion of R. typhina may be boosted with the shrinkage of A. truncatum.

4.3. AMF Inoculation Boosted the Root Growth of Both Species under Mono-Planting Condition but Weakened That of A. truncatum in Mix-Planting

Root morphological traits such as root length, root surface area, root volume, root tip and root cross have been commonly recognized to be positively associated with soil exploitation and nutrient assimilation, since the increases in these traits generally enhance the contact area of roots to soil [16,17]. Under mono-planting conditions, AMF inoculation increased the trait values in root length, root surface area, root volume, root tip and root cross of both species and thus enhanced their root contact area to soil. This observation indicates that the AMF colonization on roots stimulates the extension of root network, which provides assistance in soil nutrient assimilation and thus promotes plant growth [19,20,21]. However, the root growth of A. truncatum was prohibited under mix-planting conditions when inoculated with AMF. Since R. typhina “attracted” more AMF colonization in the resource competition for AMF, R. typhina gained more assistance from the AMF colonization in the extension of root network and nutrient assimilation. A. truncatum performed a weakened root growth with AMF inoculation under mix-planting condition. Therefore, the alien invader, R. typhina, can outcompete the native A. truncatum in the resource exploitation of belowground, which promotes the invasion of R. typhina.

5. Conclusions

Based on a manipulative experiment of alien vs. native comparison, we highlight a series of observations as: (1) the alien invader, R. typhina “attracted” more AMF colonization when facing the inter-specific competition with A. truncatum; (2) AMF colonization restricted the extension of root network for A. truncatum under mix-planting conditions compared to R. typhina; and (3) no shrinkage in body size was shown for R. typhina with nitrogen deposition compared to A. truncatum. All three observations certify that morphological traits, including both aboveground and belowground traits, determine the establishment of R. typhina. A pathway for the invasion process of R. typhina can be also speculated as: AMF colonization promotes the extension of the root network for R. typhina and thus accelerates its increase in body size. As two major components of climate change, nitrogen deposition and biological invasion bring about challenges to the sustainable development of human civilization. The present study certifies that nitrogen deposition likely weakens the performance of native species relative to the exotic invasive species, which may lead to the loss of native species and threaten the stability of local ecosystem. Future study should concern the temporal scale for the similar topic, since woody species have long vegetative growing periods, and the current study only concerns the phase of plant colonization.