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Article

Soil Microorganisms Mediated the Responses of the Plant–Soil Systems of Neotrinia splendens to Nitrogen Addition and Warming in a Desert Ecosystem

by
Zhuxin Mao
1,2,
Ming Yue
1,
Yuchao Wang
1,
Lijuan Li
1 and
Yang Li
1,2,*
1
Xi’an Botanical Garden of Shaanxi Province, Institute of Botany of Shaanxi Province, Xi’an 710061, China
2
Shaanxi Engineering Research Centre for Conservation and Utilization of Botanical Resources, Xi’an 710061, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(1), 132; https://doi.org/10.3390/agronomy14010132
Submission received: 15 December 2023 / Revised: 27 December 2023 / Accepted: 29 December 2023 / Published: 4 January 2024
(This article belongs to the Special Issue The Response of Grassland Ecosystem to Nutrient Additions)
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Abstract

:
Covering about 30% of the global total land area, desert ecosystems have been influenced by warming and nitrogen deposition. However, it remains unclear how desert ecosystems respond to warming and nitrogen deposition. Therefore, we conducted a greenhouse experiment to examine the impacts of N addition and warming on the plant–soil system of Neotrinia splendens, the dominant plant in the desert ecosystem in Northern China. Our findings revealed that low-N dose (N1) and high-N dose additions (N2) increased the biomass by 22.83% and 54.23%, respectively; meanwhile, moderate warming (T2) and severe warming (T3) decreased the biomass by 39.07% and 45.47%, respectively. N addition did not significantly affect the C:N:P stoichiometry in the plant–soil system. T2 and T3 decreased the leaf N content by 17.50% and 16.20%, respectively, and decreased the leaf P content by 10.61% and 45.29%, respectively. This resulted in the plant C:N ratio, C:P ratio, and N:P ratio increasing with warming. Furthermore, warming or N addition not only decreased soil microbial diversity, but also inhibited microbial genera associated with nutrient cycling, such as that of Tumebacillus spp., Bacillus spp., and Mortierella spp.; it additionally influenced important bacterial functions, such as nitrate reduction and ureolysis. Moreover, warming and N addition induced P limitation in the plant–soil system by inhibiting soil microorganisms, such as Mortierella spp. and Bacillus spp., which are associated with P transformation; this was also brought about by increasing the effects of leaf P content on leaf N:P. In conclusion, our results showed that warming and N addition had significant effects on the C:N:P stoichiometry of the plant–soil system through microbial mediation and led to P limitation in the system, regardless of how they affected biomass. Soil microorganisms could mediate the impacts of environmental changes on the plant–soil system. Our findings may provide valuable insights for adjusting vegetation restoration strategies in desert ecosystems under environmental changes.

1. Introduction

The desert ecosystem is an important component of terrestrial ecosystems, covering about 30% of the total global land area [1]. Like other terrestrial ecosystems, desert ecosystems have been influenced by significant human-induced environmental changes, including warming and nitrogen (N) deposition [2]. In addition to average temperature increases, extreme temperature events have become more frequent in desert ecosystems. For example, extreme high temperatures could reach 43 °C in the desert regions of Northern China [3]. N deposition is increasing in arid and semiarid regions due to rapid industrial and agricultural activities [4,5]. It has been reported that the desert ecosystem in Northern China is experiencing more severe atmospheric N deposition [5,6]. However, the impacts of warming and N deposition on desert ecosystems remain unclear.
Previous studies have shown that warming and N deposition could directly and indirectly affect plant growth. For example, increasing temperatures have been found to not only directly affect the metabolic processes of plant [7], but also indirectly affect plant growth by affecting microbial activity and nutrient availability in soil [8]. N addition could directly affect plant growth by stimulating N availability in soil [9]; meanwhile, it could indirectly affect plant growth by inducing soil acidification [10], altering microbe-mediated N transformations [9], and altering P availability in soil [7]. Furthermore, changes in C may occur in soil through alteration of plant growth under environmental changes, which, in turn could change the nutrient availability in soil or microbial and chemical rhizosphere environments [11]. Soil microorganisms maintain the turnover of matter and energy in the biosphere through the transformation of soil organic matter, nutrients, and most key soil processes [12]. Therefore, the response of soil microorganisms to environmental changes could affect the cycling of the elements in the ecosystem. For example, mycorrhizal fungi could alter plant C:N:P stoichiometry through increasing the plant N and P uptake and obtaining C from the host plant [13]. Moreover, soil microorganisms could be affected by C:N:P stoichiometry of the ecosystem. For example, a plant litter with low C:N ratio was beneficial to the bacteria in soil, while a litter with high C:N ratio was beneficial to the fungi in soil [14]. Furthermore, soil microbial diversity could alter soil nutrient availability and further affect plant growth and C:N:P stoichiometry [15]. These studies highlighted that the plant–soil system, as a whole, responded to environmental changes, while soil microorganisms may mediate elements cycling between the soil–plant–atmosphere continuum and had important effects in driving nutrient transformations and regulating ecosystem functions [16,17].
The nutrient cycling in desert ecosystems is, indeed, highly susceptible to environmental changes. Firstly, some plants in the desert are surviving at their upper biological limits [18]. Environmental changes could significantly alter plant performance and even lead to rapid species losses [19,20]. Secondly, environmental changes could significantly influence desert ecosystems by changing soil nutrient availability [21,22], because deserts are highly nutrient-limited ecosystems [23]. Thirdly, desert soil microbial communities are taxonomically distinct from other biomes and exhibit lower functional diversity with respect to nutrient cycling [24]. However, there is a lack of studies investigating the relationships of the C:N:P stoichiometry in the plant–soil system in desert ecosystems specifically. Additionally, further exploration of the pathways by which environmental changes impact desert ecosystems, particularly the dominant plants, will provide a scientific foundation for adjusting vegetation restoration strategies in these areas.
Therefore, in order to determine the effects and pathways of warming and N deposition on the desert ecosystem in Northern China, we selected the plant–soil system of Neotrinia splendens, the dominant plant, as the research object. N. splendens is distributed widely and exhibits adaptability in severe environments. Thus, it is used to protect against the wind, fixing sand in a way which assists in restoring degraded soil in China [25]. In recent years, the degradation of N. splendens vegetation has been aggravated by intensified climate change in this region (i.e., warming) and anthropic activities (i.e., intensive agriculture, fertilization, industrialization, etc.). Here, we conducted an experiment in a greenhouse to examine the effects of N addition, warming, and their interactions on the biomass, soil microorganisms, and stoichiometry of the plant–soil system of N. splendens. We hypothesized that (1) N addition, warming, and their interactions could influence the biomass and the C:N:P stoichiometries of plants and soil; (2) soil microorganisms mediate the effects of environmental changes on C:N:P stoichiometry in the plant–soil system; (3) the C:N:P stoichiometries of the plant, the soil, and the soil microorganisms play key roles in shaping plant biomass under environmental changes.

2. Materials and Methods

2.1. Experimental Design and Sampling

We collected seeds of N. splendens on the Loess Plateau in Shaanxi Province, China, in August 2021. The collected seeds were mixed together and were air-dried outside. While collecting the seeds, we excavated about 50 cm × 50 cm × 50 cm of soil from each population and brought it back to the Xi’an botanical garden in Shaanxi Province, China (109°03′ N, 34°21′ E). After removing roots and rocks, the soil collected from each population was thoroughly mixed. We put the soil in pots and placed them outside.
The experiment was performed in a greenhouse at Xi’an botanical garden in May 2022. First, seeds were germinated in trays in a greenhouse. After 15 days, the seedlings were transplanted into pots according to the design. There was one seedling in each pot to avoid competition. The experimental design was a completely randomized design with three nitrogen levels and three temperature levels. There were three replications. Overall, we used 27 pots (24 cm diameter, 17.5 cm height). Each pot was filled with 2.5 kg soil which was collected from the N. splendens population, as mentioned above.
We added N in the form of urea in early August 2022. Urea with 46% nitrogen was used as nitrogen fertilizer and was applied at three levels (N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1). The low-dose N enrichment level (N1—85 kg ha−1 yr−1) covered the current actual N input rate in the study area, in accordance with previous research [26,27], whereas the high-dose N enrichment level (N2—170 kg ha−1 yr−1) was set to simulate the response of desert ecosystems to future extreme conditions [28]. Three temperature treatment levels (T1—simulating in situ temperature, 18–23 °C; T2, 25–30 °C; T3, 32–37 °C) began in late August 2022. In order to ensure a warming effect in the greenhouse, the temperature increase in our experiment was greater than the actual situation. Moreover, T3 stimulated future extreme weather. Throughout the growing season, the soil samples were given sufficient water to ensure the survival and growth of the seedlings.
We harvested the plants on 28 September 2022. We broke the pots and separated the plants from the soil. We shook the soil off the plants as much as possible. The seedlings were washed with deionized water and then divided into the aboveground and belowground organs. The plant samples were heated at 105 °C for 30 min and then dried at 75 °C to constant weight [29]. The total biomass was the sum of the weights of the above- and belowground organs and measured using an electronic balance. The dried aboveground and belowground organs were ground and passed through a 40-mesh nylon screen, respectively. Then, a 100 g fresh soil sample was collected from each pot to measure the soil’s microbial diversity, and 200 g of the air-dried soil samples from each pot was ground, passed through a 40-mesh nylon screen, and stored in the dark at a low temperature for soil physicochemical analysis.

2.2. Plant and Soil C, N, and P Measurements

The total carbon (C) and total nitrogen (N) in the plant or the soil were determined through the PE2400II elemental analyzer (PE, USA). The total phosphorus (P) in plant samples was determined through the ICP-OES (Perkin Elmer Optima 7300 DV) after being made into ash residue at 450 °C and then dissolved in a diluted nitric acid [30].

2.3. Determination of Soil Microbial Diversity and Functions

In this study, DNA was extracted from soil samples (0.5 g) in triplicate using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). The V4-V5 region of bacterial 16S rRNA genes was amplified using the primer pairs 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) [31] through PCR using an ABI GeneAmp® 9700 Polymerase chain reaction (PCR) thermocycler (ABI, Foster, CA, USA). PCR reactions were performed in a 20 μL volume, containing 5× TransStart FastPfu buffer (4 μL), 2.5 mM dNTPs (2 μL), 5 μM of each primer (0.8 μL), TransStart FastPfu DNA polymerase (0.4 μL), template DNA (10 ng), and double-distilled water (10 μL). The PCR thermal cycling program consisted of an initial denaturation step at 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, and a final extension step at 72 °C for 10 min. The reaction was then terminated at 4 °C. PCR products were isolated from a 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), and quantified using the Quantus™ Fluorometer (Promega, Maddison, WI, USA).
In our study, we amplified the fungal ITS1 region using the ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCATCGATGC-3′) primers [31]. Additionally, we carried out bacterial 16S rRNA gene amplification. The amplicons were purified in equal amounts and paired-end sequenced (2 × 300 bp) using the Illumina MiSeq platform (Illumina), following standard protocols provided by Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China. The raw reads obtained from sequencing were deposited into the NCBI Sequence Read Archive (SRA).
To analyze the Illumina sequencing data, we used Flash with the option “max-overlap 200” to merge paired-end reads, and any unassembled sequences were removed. QIIME 1.9.1 was employed to process and analyze the FASTQ files. Bacterial sequences were searched against the Ribosomal Database Project Classifier, and chimeric sequences were identified and discarded. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (version 7.1). The taxonomy of each OTU representative sequence was determined through comparing it against the 16S rRNA database (Silva) with a confidence threshold of 0.7. Fungal ITS sequences were assigned to taxa using the naive Bayesian classifier. OTUs were also clustered with a 97% similarity cutoff for the fungal dataset. OTUs with low abundance were filtered out using the OTU table [31].
The Shannon–Wiener index was selected to characterize α diversity of bacterial and fungal communities and was calculated using the following formula:
Shannon–Wiener index:
H = − ∑ i = 1 n P i ln P i
where Pi is the proportional abundance of species i [32].
FAPROTAX [33] was utilized for assessing the functional potential of the communities.The FAPROTAX database, which provides functional annotation of prokaryotic taxa, was employed to predict biogeochemical cycles or ecological functions [34]. To estimate the relative species, the resampled OTU table and OTU taxonomy information were aligned with the Silva database. This alignment allowed for the summarization of functional classification and annotation information [33]. Subsequently, the resampled OTU table and OTU classification were subjected to blasting and annotation using the Silva database. The annotation results were then downloaded and analyzed based on the functional categories. The analysis results were represented using a heat map. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession numbers can be found here: BioProject, PRJNA1010112.

2.4. Data Analysis

All data were log-transformed to reach the model assumptions if necessary, and all the stoichiometric ratios reported in our study were mass ratios. Two-way ANOVAs were used to test N-addition, warming, and their interactions on plant biomass, plant C:N:P stoichiometry, soil C:N:P stoichiometry, soil microbial diversity, and soil microbial functions, respectively. Furthermore, we selected genera of fungi and bacteria that were significantly and marginally significantly affected by N addition or warming (p < 0.10), and explored their correlations with C:N:P stoichiometry in the plant–soil system, respectively. Meanwhile, redundancy analysis (RDA) was used to confirm the major microbial drivers of C:N:P stoichiometry of the plant–soil system. In addition, the comprehensive influences of environmental changes (N addition, warming), plant C:N:P stoichiometry, soil C:N:P stoichiometry and soil microbial diversity on the plant biomass were analyzed via principal component analysis (PCA) and variance partitioning (VPA, “vegan” package in R). To avoid multicollinearity and overfitting, the variable using a forward selection method was selected before analysis [6]. Finally, structural equation modeling (SEM) was performed to further examine the direct and indirect effects linking environmental changes and plant biomass through plant C:N:P stoichiometry, soil C:N:P stoichiometry, and soil microbial diversity. SEM was conducted with Amos software (v 24.0, IBM, New York, NY, USA), and the best quality of fits was evaluated with the χ2 test (p > 0.05), CFI (comparative fit index) value (CFI > 0.9), and low RMSEA (root mean square error of approximation) value (RMSEA < 0.08). According to the analysis results of VPA and SEM, the Venn diagram and path diagram were drawn using Microsoft Office PowerPoint 2016. The heat map was created using the R package pheatmap. Other figures were created using GraphPad Prism 9.

3. Results

3.1. Plant Growth

According to the results of the two-way ANOVAs, N addition, warming, and their interactions all had significant effects on the total biomass and the biomass allocation of N. splendens (all p < 0.05, Figure 1a–c, Table S1). For the main effects, N addition stimulated the aboveground biomass, the belowground biomass, and the overall biomass, while warming decreased them. Specifically, N2 and N3 stimulated aboveground biomass by 26.16% and 48.76%, respectively, stimulated belowground biomass by 17.03% and 63.75% respectively, and stimulated overall biomass by 22.83% and 54.23%, respectively; T2 and T3 decreased aboveground biomass by 36.26% and 43.68%, respectively, decreased belowground biomass by 43.63% and 48.36%, respectively, and decreased overall biomass by 39.07% and 45.47%, respectively. Moreover, the interaction of N addition and warming had significant effects on the aboveground biomass (p = 0.03, Table S1).

3.2. The C:N:P Stoichiometry of the Plant

According to the results of two-way ANOVAs, N addition had no significant effects on the C, N, or P stoichiometries of N. splendens (all p > 0.05, Figure 2a–l). Warming had significant effects on the C, N, and P stoichiometries (all p < 0.05, Figure 2b,c,e–j,l), except the C content and the root C:P (all p > 0.05, Figure 2a,d,k). T2 and T3 significantly decreased the leaf N content by 17.50% and 16.20%, respectively, and significantly decreased the leaf P content by 10.61% and 45.29%, respectively. T2 and T3 significantly changed the root N content by −14.06% and 10.85%, respectively, and significantly decreased the root P content by 26.34% and 29.95%, respectively. Thus, T2 and T3 significantly increased the leaf C:N ratio by 20.39% and 18.30%, respectively (p = 0.03, Figure 2g); increased the leaf C:P ratio by 10.28% and 72.31%, respectively (p = 0.01, Figure 2h); changed the leaf N:P ratio by −9.72% and 45.77%, respectively (p = 0.01, Figure 2i); changed the root C:N ratio by 28.37% and −0.46%, respectively (p < 0.05, Figure 2j); increased the root N:P ratio by 0.87% and 40.80%, respectively (p = 0.03, Figure 2l). Moreover, N addition and warming had significant interactive effects on the root P content and the root C:N:P ratios (p < 0.05, Figure 2f,i–l).

3.3. The C:N:P Stoichiometry of the Soil

According to the results of the two-way ANOVAs, warming had significant effects on the P content in soil (p < 0.05, Figure 3c). T2 and T3 significantly changed the soil P content by 14.34% and decreased it by 0.97%, respectively. Moreover, N addition and warming had a significant interactive effect on the soil C content (p = 0.04, Figure 3a). N addition, warming, and their interactions had no significant effects on the soil C:N:P ratios (all p > 0.05, Figure 3d–f).

3.4. Soil Microbial Diversity and Function

Although N addition had no significant effects on soil bacterial diversity (p > 0.05, Figure 4a), it significantly decreased the relative abundance of Bacillus (p = 0.03, Figure 5a, Table 1), and significantly decreased bacterial functions, such as chemoheterotrophy, aerobic chemoheterotrophy, nitrate reduction, ureolysis, chitinolysis, hydrocarbon degradation, dark hydrogen oxidation, and aromatic hydrocarbon degradation (all p < 0.05, Table 2). Warming had significant effects on soil bacterial diversity (p = 0.04, Figure 4a). T2 and T3 decreased the Shannon–Wiener diversity index by 0.87% and 1.79%, respectively. Furthermore, warming significantly influenced the relative abundance of Gaiella, Bacillus, and Dongia (all p < 0.05, Figure 5a, Table 1) and influenced bacterial functions, such as nitrate reduction, fermentation, photoautotrophy, oxygenic photoautotrophy, hydrocarbon degradation, methylotrophy, aromatic hydrocarbon degradation, methanol oxidation, and methanotrophy (all p < 0.05, Table 2). There were no interactions between N addition and warming on bacterial functions (all p > 0.05, Table 2).
According to the results of two-way ANOVAs, N addition, warming, and their interactions all played significant roles on soil fungal diversity (all p < 0.05, Figure 4b). N3 decreased the Shannon–Wiener diversity index by 7.67%, while T3 decreased it by 5.78%. In addition to the main effect, the interaction between N addition and warming significantly decreased the Shannon–Wiener diversity index of soil fungi. From the genus, N addition significantly decreased the relative abundance of Neocosmospora; while warming significantly influenced the relative abundance of Gibellulopsis, Stachybotrys, Funneliformis, Cladosporium, and Alternaria (all p < 0.05, Figure 5b, Table 1). From the fungal function, N addition and warming significantly decreased pathotroph, saprotroph, pathotroph–saprotroph, and saprotroph–symbiotroph, respectively (all p < 0.05, Table 2). There were no interactions between N addition and warming on fungal functions (all p > 0.05, Table 2).

3.5. Correlations between Soil Microorganisms and C:N:P Stoichiometry in the Plant–Soil System

For bacteria, the relative abundances of Gaiella, Bacillus, Dongia, and Tumebacillus were positively correlated with the soil C:N ratio; the relative abundance of Gaiella was positively correlated with soil C content; the relative abundance of Tumebacillus was negatively correlated with soil N content. The relative abundance of Bacillus was significantly correlated with root P content, leaf C:P ratio, and leaf N:P ratio, respectively (all p < 0.05, Figure 6a). For fungi, the relative abundance of Funneliformis positively correlated with soil P content; since the relative abundances of Emmonsiellopsis and Lophotrichus were negatively correlated with soil N content, they were negatively correlated with soil C:N ratio and positively correlated with soil N:P ratio (p < 0.05, Figure 6a). The relative abundances of Cladosporium and Emmonsiellopsis were positively correlated with P content in the leaf and root, respectively (p < 0.05, Figure 6a). Root N content was negatively correlated with the relative abundances of Mortierella and Cladosporium (all p < 0.05, Figure 6a). Root P content was positively correlated with the relative abundance of Emmonsiellopsis (p < 0.05, Figure 6a). Leaf N:P ratio was negatively correlated with the relative abundances of Mortierella, Neocosmospora, Gibellulopsis, Cladosporium, and Emmonsiellopsis, while the root C:N ratio was positively correlated with the relative abundance of Mortierella and Gibellulopsis (all p < 0.05, Figure 6a). The results of RDA were consistent with the correlations (Figure 6b).

3.6. The Relative Contributions of Different Factors to Plant Growth

The results from principal component analysis (PCA) showed that the cumulative contribution rate of the two principal components was 47.92%; here, principal component 1 contributed 26.42% and principal component 2 contributed 21.50%. Principal component 1 mainly contained the C:N:P stoichiometry of the leaf, while principal component 2 mainly contained the C:N:P stoichiometry of the root (Figure 7a).
The variation partitioning (VPA) results revealed that environmental factors, plant stoichiometry, soil stoichiometry, and soil microbial diversity could explain the variation in biomass by 13.98%, 58.93%, 2.30%, and 3.95%, respectively (Figure 7b). Moreover, the interactions between soil microbial diversity and plant stoichiometry, and the interactions between soil microbial diversity, plant stoichiometry, and environmental factors, could explain the variation in biomass by 9.08% and 9.32%, respectively. Similarly, the interactions between soil stoichiometry, plant stoichiometry, and soil microbial diversity, and the interactions between soil stoichiometry, soil microbial diversity, and environmental factors, could explain the variation in biomass by 3.66% and 4.46%, respectively (Figure 7b).
The final SEMs fit the data well and further demonstrated the driving mechanisms of stoichiometry in the plant–soil system to plant growth under N addition and warming (Figure 8). N addition and leaf N:P ratio (N:Pl) directly affected plant biomass, and the model explained 42% of the variation in biomass. Specifically, N addition had positive effects on plant biomass (p < 0.001), while N:Pl had negative effects on plant biomass (p < 0.05). The soil N:P ratio had positive effects on root N:P ratio (p < 0.010), while the root N:P ratio had positive effects on N:P in the leaf (p < 0.05).

4. Discussion

Our results demonstrated that warming, N addition, and their interactions all had significant effects on the biomass of N. splendens; meanwhile, the effects of warming on the stoichiometry in the plant–soil system of N. splendens were stronger than those of N addition. N addition and warming could influence C:N:P stoichiometry in the plant–soil system through decreased soil microbial diversity and functions, and they ultimately affected the biomass accumulation of N. splendens. With N deposition and warming, the plant–soil system would become limited by available P content in soil.

4.1. Impacts of Environmental Changes on Biomass and Stoichiometry of the Plant–Soil System

According to our results, plant C:N:P stoichiometry could explain 58.93% of the variation in plant biomass. This finding suggested that plant stoichiometry was closely associated with growth, metabolism, and nutrient absorption and utilization [35]. Additionally, our results showed that both N addition and warming led to an increase in plant N:P. Combined with the results of PCA and SEM, it was shown that plants were limited by P with N addition or warming in our study. This is consistent with previous findings from ecosystems at the global scale [36,37,38]. However, N addition and warming affected the C:N:P stoichiometry in the plant–soil system of N. splendens via different ways in our study.
Contrary to our hypothesis, N addition had a limited significant effect on the stoichiometry of the plant–soil system. This finding aligned with a previous study that found no changes in the C:N:P stoichiometry of the plant with N addition [32]. Despite the verification that N addition could increase the soil-available N for plants [39,40,41], we observed no changes in the soil N content. Meanwhile, considering that the soil was given sufficient water, there was enough soil water to have accelerated mineralization of soil organic matter. Thus, our results may suggest N loss from excessive N application [42]. This was confirmed further by the findings that N addition may have a limited impact on soil P content according to our results. Due to the coupling of major elements in various biochemical and cellular constituents [43], plants absorbed N and P proportionally from the soil. Remarkably, the P absorbed by plants concomitantly with the additional N may be relatively minor. Furthermore, the low content of available P in soil would also limit the absorption of N by plants, and, in this study, it eventually led to excessive N loss. Thus, these pathways may result in N addition having no significant effects on soil N and P stoichiometry.
Consistent with our hypothesis and previous research, warming had significant effects on the C:N:P stoichiometry of plants [44]. Firstly, warming led to a significant decrease in leaf N and P content, which indicated a lower photosynthetic rate, a slower growth rate, and a weaker resource competitive ability [45,46]; these consequently led to a decrease in biomass accumulation. Our findings were consistent with previous reports demonstrating that plant N and P contents declined because of warming [47]. This was attributed to the fact that warming could stimulate the rates of biochemical reactions catalyzed by N-rich enzymes and P-rich RNA contents in plants, causing the plants to allocate more nutrients to other molecules [48]. Secondly, warming decelerated the soil microbial turnover rate [49], which, in turn, reduced the available N and P in the soil. Because N and P in potting soil became unavailable with warming, the amounts of N and P left in the pot increased. Thus, there was a seemingly contradictory result that warming increased the soil total P content. Furthermore, the increase in plant C:N:P stoichiometry further proved that the soil-available N and P were reduced in our study. Therefore, warming had a significant influence on the C:N:P stoichiometry of the plant–soil system through decreasing plant physiological activities and soil-available nutrients in our study.

4.2. Soil Microorganisms Mediated the Impacts of Environmental Changes

Combing the path analysis in SEM and the correlations between soil microorganisms and the C:N:P stoichiometry in the plant–soil system, our VPA results revealed that the interactions between soil microbial diversity and the other three factors could explain the greater variations in biomass. This suggested that soil microorganisms played a mediating role in the response of the plant–soil system to environmental changes. Soil microbial diversity was generally considered to be closely related to soil nutrient availability, as higher diversity in a microbial community could enlarge the sources of nutrient acquisition and stimulate the decomposition of recalcitrant organic matter [15]. Thus, previous studies and our study both found that environmental changes, particularly warming, could reduce soil nutrient availability by decreasing soil microbial taxonomic diversity, ultimately influencing the C:N:P stoichiometry and plant growth [50,51].
In our study, we not only examined the impacts of environmental changes on microbial taxonomic diversity but also further analyzed the microbial genus and function (Figure 4, Figure 5 and Figure S1), as well as the relationships between them and the C:N:P stoichiometry in the plant–soil system. The comprehensive approach better elucidated the mediating roles of soil microorganisms in the response of plant–soil systems to environmental changes.
Firstly, for the C cycle, warming reduced C inputs due to a significant decline in root biomass [52]. In a C-limited environment, bacteria could utilize scarce resources. For instance, soil bacteria took advantage of one-carbon compounds such as methanol or methane for energy [53]. This was supported by the increased processes of methanol oxidation and methylotrophy with warming in our study. This could also be proved by the fact that warming increased activity in converting carbohydrates to alcohol or organic acids by stimulating fermentation in our study. Secondly, for the N cycle, we found that N addition decreased ammonia oxidation, which was the rate-limiting step of nitrification [53]. This observation was supported by the fact that low-dose N addition reduced bacterial nitrification in our study. Furthermore, low-dose N addition increased nitrate reduction, which could induce denitrification and result in nitrogen loss [54]. Warming could affect the soil N cycle by inhibiting the relative abundance of Tumebacillus, which was strongly associated with nitrate reduction in soil [55], and by inhibiting the bacteria’s nitrate reduction function in our study. Together, they resulted in a decrease in available N in the soil with warming in the soil in our study. Thirdly, for the P cycle, according to fungal functions in our study, warming could inhibit the relative abundance of Mortierella and Bacillus, which are known for their ability to dissolve soil P [56,57], as well as saprophytic fungi, which could play a key role in plant residue decomposition and nutrient cycling [58]. Although warming increased the soil total P content, the decreases in plant P content indicated that warming decreased the soil-available P content by limiting P transformation in soil [59].
In general, our results indicated that environmental changes could influence soil nutrient availability through affecting soil microbial function and diversity, which could impact plant nutrient content consequently and, ultimately, affect plant biomass accumulation. Soil microorganisms played a mediating role in the response of the plant–soil system to environmental changes. Our results were consistent with the temperature–biogeochemistry hypothesis, which suggested that temperature altered the plant stoichiometry through the effects on the soil microorganisms [48].

4.3. Soil-Available P Content May Become a Limiting Factor

Although our study found that soil stoichiometry explained little of the variation in biomass, it did not imply that soil stoichiometry was unimportant. This may be attributed to the fact that we used total nutrients rather than available nutrients in calculating soil C:N:P stoichiometry in our study. Soil-available nutrient stoichiometry may be highly correlated with plant stoichiometry and could influence biomass accumulation [59], while total nutrient elements in the soil may not be correlated with plant stoichiometry, because they contain forms that cannot be readily absorbed by plants [60], especially when soil microorganisms are suppressed. It is generally considered that the available nutrient content of arid desert soil we used in this study is low. Therefore, the total soil nutrients could contribute minimally to the plant biomass accumulation.
Our results indicated that soil-available P content rather than the total P content may be the limiting factor in the plant–soil system. This could be supported by the following findings in our experiment: Firstly, our results suggested that N addition and warming might intensify the limiting effect of P on plant growth [61,62] due to the decrease in soil P availability through microbe-mediated P transformation [32,63]. Our results showed that high-dose N addition and warming inhibited the relative abundance of Mortierella and Bacillus, both of which had the abilities to dissolve soil P and increased soil-available P content [56,57]. As a result, high-dose N addition and warming could lead to a decrease in soil-available P content, potentially becoming a limiting factor in the plant–soil system. Secondly, the leaf N:P ratio was considered to be an indicator of nutrient supply for plant growth [36,37,38]. We found that the effect of plant P content on the plant N:P ratio (4.73, p < 0.001) was greater than that of plant N content (0.34, p > 0.05) (Figure S2), indicating that the changes in the N:P ratio were primarily determined through plant P content [64]. Plant P content was closely related to soil-available P content [59]. Therefore, our results were consistent with previous research, suggesting that environmental changes could induce P limitation [61,62,65], while soil microorganisms may mediate the limitation [66].

5. Conclusions

Our results partially confirm the hypotheses that warming, N addition, and their interactions significantly influence the biomass of N. splendens and that warming and its interactions with N addition significantly influence the C:N:P stoichiometry of the plant–soil system. Environmental changes and the plant stoichiometry were found to explain a larger proportion of the biomass variation, while soil microorganisms explained biomass variation through their interactions with other factors. Furthermore, warming and N addition induced the system into P limitation by inhibiting the soil microorganisms associated with P transformation and by increasing the effects of leaf P content on leaf N:P. Our findings indicate that soil microorganisms mediated the effects of N addition and warming on the plant–soil systems of N. splendens.
N. splendens is one of the main plants used for ecological restoration in the desert ecosystem of Northern China. Our results demonstrate that, in the context of warming and nitrogen deposition, we should take the changes in limiting factors and the soil microbial community structure into account in the plant–soil systems of N. splendens. However, our experiment was conducted under controlled greenhouse conditions. The growing conditions in a greenhouse are different from those in the wild. Our results theoretically revealed the possible pathways by which warming and N deposition may affect the plant–soil system of N. splendens. In order to develop corresponding countermeasures to ensure the stability of desert ecosystem structure and functions, our results need to undergo a validation study under natural field conditions in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14010132/s1, Figure S1: N addition–warming treatments induced changes in soil microbial β-diversity (a) Bacteria (b) Fungi. Figure S2: The relationships between leaf N:P and leaf N content or leaf P content. Table S1: N addition (N), warming (W) and their interactions (N*W) on biomass and the C:N:P stoichiometry of the plant–soil system.

Author Contributions

Conceptualization, Z.M., M.Y. and Y.L; Data curation, Y.L; Investigation, Z.M., Y.W. and L.L.; Methodology, Z.M., M.Y., Y.W. and Y.L; Writing—original draft preparation, Z.M. and Y.L.; Writing—review and editing, M.Y. and Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by “the Natural Science Foundation of Shaanxi Province of China [2021JM-597, 2020ZDLSF06-01, 2022JM-132]”, “the Scientific and Technological Innovation Project of Shaanxi Forestry Academy of Sciences [SXLK2021-0204]”, “Key Project of Shaanxi Academy of Sciences [2021K-18]”, and “The project of the first investigation of wild plants resources in Xi’an [KRDL K6-2207039]”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We fully appreciate the editors and all anonymous reviewers for their constructive comments on this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. N addition–warming treatments induced changes in (a) aboveground biomass, (b) belowground biomass, and (c) total biomass of N. splendens. Values were means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences in biomass among N addition–warming treatments.
Figure 1. N addition–warming treatments induced changes in (a) aboveground biomass, (b) belowground biomass, and (c) total biomass of N. splendens. Values were means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences in biomass among N addition–warming treatments.
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Figure 2. N addition–warming treatments induced changes in the C:N:P stoichiometry of the plant ((a): Leaf C content; (b): Leaf N content; (c): Leaf P content; (d): Root C content; (e): Root N content; (f): Root P content; (g): Leaf C:N ratio; (h): Leaf C:P ratio; (i): Leaf N:P ratio; (j): Root C:N ratio; (k): Root C:P ratio; (l): Root N:P ratio). Values were means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences among N addition–warming treatments. See Figure 1 for abbreviations.
Figure 2. N addition–warming treatments induced changes in the C:N:P stoichiometry of the plant ((a): Leaf C content; (b): Leaf N content; (c): Leaf P content; (d): Root C content; (e): Root N content; (f): Root P content; (g): Leaf C:N ratio; (h): Leaf C:P ratio; (i): Leaf N:P ratio; (j): Root C:N ratio; (k): Root C:P ratio; (l): Root N:P ratio). Values were means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences among N addition–warming treatments. See Figure 1 for abbreviations.
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Figure 3. N addition–warming treatments induced changes in C:N:P stoichiometry of the soil ((a): Soil C content; (b): Soil N content; (c): Soil P content; (d): Soil C:N ratio; (e): Soil C:P ratio; (f): Soil N:P ratio). Values were means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences among N addition–warming treatments. See Figure 1 for abbreviations.
Figure 3. N addition–warming treatments induced changes in C:N:P stoichiometry of the soil ((a): Soil C content; (b): Soil N content; (c): Soil P content; (d): Soil C:N ratio; (e): Soil C:P ratio; (f): Soil N:P ratio). Values were means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences among N addition–warming treatments. See Figure 1 for abbreviations.
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Figure 4. N addition–warming treatments induced changes in (a) bacterial and (b) fungal soil microbial diversity. Values are means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences among N addition–warming treatments. See Figure 1 for abbreviations.
Figure 4. N addition–warming treatments induced changes in (a) bacterial and (b) fungal soil microbial diversity. Values are means ± S.E., n = 3 for each treatment. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C. Letters showed the significant differences among N addition–warming treatments. See Figure 1 for abbreviations.
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Figure 5. N addition–warming treatments induced changes in relative abundance of bacterial (a) and fungal (b) community composition at the genus level. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C.
Figure 5. N addition–warming treatments induced changes in relative abundance of bacterial (a) and fungal (b) community composition at the genus level. N0—no urea; N1—85 kg ha−1; N2—170 kg ha−1. T1—simulating in situ temperature, 18–23 °C; T2—moderate warming, 25–30 °C; T3—severe warming, 32–37 °C.
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Figure 6. (a) The correlations between soil microbial genus with C:N:P stoichiometry of the plant–soil system. (b) RDA ordination diagram displaying the major microbial drivers of C:N:P stoichiometry of the plant–soil system. Green—bacteria; red—fungi.
Figure 6. (a) The correlations between soil microbial genus with C:N:P stoichiometry of the plant–soil system. (b) RDA ordination diagram displaying the major microbial drivers of C:N:P stoichiometry of the plant–soil system. Green—bacteria; red—fungi.
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Figure 7. (a) PCA ordination diagram displaying the major drivers in the plant–soil system of N. splendens. C, N, and P represented the content of C, N, and P in the leaf, the root, or the soil, respectively; CN, CP, and NP represented the C:N ratio, the C:P ratio, and the N:P ratio in the leaf, the root, or the soil, respectively. (b) Variation partitioning in biomass.
Figure 7. (a) PCA ordination diagram displaying the major drivers in the plant–soil system of N. splendens. C, N, and P represented the content of C, N, and P in the leaf, the root, or the soil, respectively; CN, CP, and NP represented the C:N ratio, the C:P ratio, and the N:P ratio in the leaf, the root, or the soil, respectively. (b) Variation partitioning in biomass.
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Figure 8. Pathway model of the multivariate effects of N addition, warming, soil microbial diversity, soil C:N:P stoichiometry, and plant C:N:P stoichiometry on biomass of N. splendens. Solid red arrows indicate significantly negative relationships. Solid blue arrows represent significantly positive relationships. Dashed lines (both red and blue) indicate that paths are insignificant. Numbers connected arrows are standardized path coefficients (^ p < 0.10; * p < 0.05; ** p < 0.01). R2 values represent the percentages of the variance explained by the model.
Figure 8. Pathway model of the multivariate effects of N addition, warming, soil microbial diversity, soil C:N:P stoichiometry, and plant C:N:P stoichiometry on biomass of N. splendens. Solid red arrows indicate significantly negative relationships. Solid blue arrows represent significantly positive relationships. Dashed lines (both red and blue) indicate that paths are insignificant. Numbers connected arrows are standardized path coefficients (^ p < 0.10; * p < 0.05; ** p < 0.01). R2 values represent the percentages of the variance explained by the model.
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Table 1. The effects of N addition (N), warming (W), and their interactions (N*W) on the relative abundance of soil microorganisms.
Table 1. The effects of N addition (N), warming (W), and their interactions (N*W) on the relative abundance of soil microorganisms.
GenusTreatments
NWN*W
FpFpFp
BacteriaGaiella0.270.774.660.026.210.00
Bacillus4.200.0312.97<0.0013.330.03
Blastococcus0.550.590.690.513.350.03
Dongia1.160.344.690.032.520.08
FungiNeocosmospora3.950.040.430.660.970.45
Gibellulopsis0.310.7411.53<0.0010.410.80
Scytalidium2.090.103.790.142.950.72
Stachybotrys2.090.153.790.042.950.05
Funneliformis3.030.0711.64<0.0012.400.09
Pseudallescheria1.900.180.850.445.030.01
Cladosporium0.420.678.550.001.140.37
Alternaria1.200.325.940.011.380.28
Table 2. The effects of N addition (N), warming (W), and their interactions (N*W) on soil microbial function.
Table 2. The effects of N addition (N), warming (W), and their interactions (N*W) on soil microbial function.
Treatments
FunctionNWN*W
FpFpFp
BacteriaUreolysis8.560.000.090.910.860.51
Dark hydrogen oxidation5.980.012.480.110.980.44
Chemoheterotrophy4.310.030.730.502.420.09
Aerobic chemoheterotrophy3.750.040.490.622.370.09
Fermentation2.620.105.210.021.730.19
Nitrate reduction5.670.019.530.002.310.10
Hydrocarbon degradation5.950.015.500.011.010.43
Aromatic hydrocarbon degradation7.680.003.850.041.810.17
Chitinolysis 8.200.001.640.221.240.33
Photoautotrophy 1.880.184.670.020.340.85
Oxygenic photoautotrophy1.580.235.340.020.450.77
Methanotrophy0.980.373.610.030.180.61
Methanol oxidation0.630.555.190.020.110.98
FungiPathotroph3.860.040.190.830.440.78
Saprotroph0.960.403.460.051.360.29
Pathotroph–saprotroph4.100.031.100.351.320.30
Saprotroph–symbiotroph4.280.033.490.052.410.09
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Mao, Z.; Yue, M.; Wang, Y.; Li, L.; Li, Y. Soil Microorganisms Mediated the Responses of the Plant–Soil Systems of Neotrinia splendens to Nitrogen Addition and Warming in a Desert Ecosystem. Agronomy 2024, 14, 132. https://doi.org/10.3390/agronomy14010132

AMA Style

Mao Z, Yue M, Wang Y, Li L, Li Y. Soil Microorganisms Mediated the Responses of the Plant–Soil Systems of Neotrinia splendens to Nitrogen Addition and Warming in a Desert Ecosystem. Agronomy. 2024; 14(1):132. https://doi.org/10.3390/agronomy14010132

Chicago/Turabian Style

Mao, Zhuxin, Ming Yue, Yuchao Wang, Lijuan Li, and Yang Li. 2024. "Soil Microorganisms Mediated the Responses of the Plant–Soil Systems of Neotrinia splendens to Nitrogen Addition and Warming in a Desert Ecosystem" Agronomy 14, no. 1: 132. https://doi.org/10.3390/agronomy14010132

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