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Article

Metagenomic Insights into How Understory Vegetation Enhances Soil Nitrogen Availability via Microbial Nitrogen Transformation in Poplar Plantations

by
Wenyu Jia
1,
Tong Li
1,
Peilei Ye
1,
Yuxin Chen
1,
Ruoning Zhu
2,
Ruixin Yan
1,
Haoran Yue
1 and
Ye Tian
1,3,*
1
College of Forestry and Grassland, Nanjing Forestry University, Nanjing 210037, China
2
Third Construction Co., Ltd. of China Construction First Bureau Group, Beijing 100161, China
3
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(7), 1537; https://doi.org/10.3390/agronomy15071537
Submission received: 19 May 2025 / Revised: 19 June 2025 / Accepted: 23 June 2025 / Published: 25 June 2025
(This article belongs to the Section Soil and Plant Nutrition)
Browse Figures
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Abstract

Long-term monoculture of poplar plantations for industrial material production has been widely reported to cause severe soil degradation, while the presence of understory vegetation might enhance soil nitrogen (N) transformation and supply. This study employed a field experiment using a randomized block design with three blocks and four understory treatments, including understory removal, N-fixing species planting, single-species retention, and diverse vegetation retention, in poplar plantations on a mid-latitude alluvial plain in China over 6 years to assess the effects of different species and richness of understory on soil N transformation and related microbial traits via 15N assays and shotgun metagenomics. The results showed that understory removal significantly reduced soil N transformation rates, bacterial abundance, and gene abundance associated with N transformation. Compared to a single-species understory, retaining a diverse understory with high species richness significantly increased soil gross N transformation rate of mineralization by 149%, nitrification by 221%, and immobilization by 85%; comprehensively enriched dominant bacterial phyla; and elevated gene abundances of gdh_K15371, ureB, hao, and amoA_B associated with N transformation. No significant difference in N transformation rates existed between N-fixing species planting treatment and single-species retention treatment, while N-fixing species planting treatment specifically promoted the soil bacterial phyla Nitrospirae and Chloroflexi, and increased the gene abundances of gdh_K15371 and hao. These findings demonstrate that both introducing N-fixing species and an increase in species richness of the understory effectively promoted soil N transformation but that different underlying mechanisms existed. Planting N-fixing species selectively increased the soil bacterial phyla of Nitrospirae and Chloroflexi, whereas the increase in species richness broadly enriched soil bacterial diversity, thereby inducing the enrichment of the functional genes and enhancing soil N transformation. In conclusion, both planting N-fixing species and retaining diverse understory vegetation were effective strategies for maintaining sustainable management of poplar plantations by increasing soil N availability.

1. Introduction

Long-term monoculture and intensive management have been widely reported to cause decline in soil fertility, productivity, and ecological functions in plantation forests such as poplar (Populus spp.) and eucalyptus (Eucalyptus spp.), threatening their sustainability [1,2,3,4]. As an essential nutrient in plantation forests, nitrogen (N) supply critically regulates tree growth. Optimizing soil N availability, therefore, represents a fundamental requirement for sustainable forest management [5]. Conventional approaches to enhance soil N supply in plantations primarily include fertilization and intercropping with N-fixing tree species. However, fertilization significantly increases silvicultural costs [6], while mixing in plantations presents several technical challenges [7], thus inducing practical constraints in plantation management.
Although abundant N presents in forest soils, over 90% exists as macromolecular organic compounds that require microbial mineralization and transforming into plant-available forms (NH4+-N and NO3−-N) prior to plant uptake [8]. Therefore, enhancing N mineralization through effective stand management is essential for maintaining long-term N supply. Soil microorganisms act as primary drivers for N transformation in soils [9], precisely regulating these processes through enzyme systems encoded by functional genes [10]. Metagenomic approaches can reveal the properties of soil functional genes related to N-transformation and quantify the N transformation potential [11,12], providing critical insights for evaluating soil N supply capacity via the microbial community in plantation forests. Therefore, silvicultural practices that optimize microbial community structure to enhance the abundance of N-transformation genes and functional potential represent a key strategy for maintaining sustainable plantation productivity.
As the primary source of soil organic inputs, litter properties regulate microbial community composition and functional differentiation in soils [13]. The effects of the quantity and quality of litter on microbial community structure and nutrient cycling have been extensively evaluated [14,15]. Comparative studies on the removal and addition of litter have revealed that the reduction in litter supply significantly inhibits the abundance and diversity of soil microbial communities, decreases the rate of nitrogen transformation, and reduces soil-available N [16,17]. In contrast, the increase in litter effectively enhances microbial diversity and N transformation efficiency [16,18]. Litter quality also significantly influences soil N transformation. The introduction of high-quality litter, such as that from N-fixing plants or litter with a low C/N ratio, markedly enhances soil microbial diversity, accelerates N mineralization, and improves soil N supply [19,20,21]. Recent studies have also demonstrated that increased litter diversity provides abundant and diverse substrates for soil microorganisms and significantly regulates the soil N transformation process by influencing the structure and function of microbial communities [22]. For instance, research in mixed-species plantations has shown that the mixing of tree litter could reshape the structure of soil microbial communities and regulate N cycling [23]; however, most of the studies were generally carried out in mixed plantations with only two tree species [24,25,26] or mixed with N-fixing tree species [27,28]. It remains unclear whether further enhancements in species richness can yield better effects on soil N supply or not.
Abundant understory vegetation generally exists in plantations. Like mixing tree species, the presence of understory could also affect the microbial community and N cycling in soil through litter input [29,30]. Recently, the role of understory vegetation in soil nutrient cycling in plantations has received great attention, and most studies have proved that it is necessary to reasonably preserve understory vegetation to ensure yield and sustainability [31]. The understory vegetation usually has high species richness in plantations. However, most studies have considered understory vegetation as a whole, neglecting its species richness and the potential effects of species richness on soil nutrient cycling.
Poplar are the extensively cultivated and are high-yield industrial crops for fiber production in middle latitudes of the world [32]. With long-term monoculture, soil N depletion became severe in poplar plantations, significantly constraining the productivity [33]. Poplar plantations are usually cultivated with wide spacing, resulting in dense understory vegetation with high species diversity [34]. However, influenced by traditional management paradigms, understory vegetation has long been perceived as a competitor for soil nutrients and water resources, and thus was generally removed from the plantations. Considering the possible effects of understory vegetation and its diversity, exploring the impacts of species richness of understory vegetation on soil nutrient cycling and supply can effectively evaluate the functions of understory vegetation and provide effective information for maintaining site productivity. Therefore, we set up experimental treatments with varying understory species and species richness in a poplar plantation to evaluate their effects on soil N transformation, microbial communities, and key functional genes associated with N transformation. Two hypotheses were posited: (1) diverse understory vegetation and N-fixing species planting enhance soil N transformation by optimizing microbial community structure and increasing the abundance of functional genes related to N-cycling; (2) the underlying mechanisms driving these enhancements differ between diversified understory vegetation and N-fixing species planting.

2. Materials and Methods

2.1. Experimental Design and Sample Collection

A long-term in situ understory vegetation control experiment was conducted at Malanghu Forest Farm in Suqian City, Jiangsu Province, China (33°32′ N, 118°36′ E). This region belongs to a semi-humid climate zone, characterized by a mean annual temperature of 14.4 °C and an average annual precipitation of 910 mm [30]. The soil is yellow-brown soil developed from alluvial deposits of Hongze Lake, which correspond to Albi-Udic Argosols based on the Chinese Soil Taxonomy (CST) classification system, or Hortic Umbrisols according to World Reference Base for Soil Resources (WRB) classification. The soil’s basic properties are as follows: the pH value is 6.65, the bulk density is 1.13 g·cm−3, and the organic carbon and total N contents are 25.66 g·kg−1 and 2.49 g·kg−1, respectively [30].
The experimental plantation was established in March 2016 using one-year-old rooted cuttings of P. deltoides clone ‘Nanlin-3804’, arranged with a spacing of 6 m × 6 m. An understory vegetation survey conducted in July 2017 found no shrubs in the understory vegetation, and the dominant herbaceous species included Echinochloa crusgalli, Setcreasea purpurea, Rhizoma cyperi, and Ammannia coccinea, representing approximately 60%, 20%, 10%, and 10% of the total coverage [30].
Four types of understory vegetation treatments were set up, including understory removal (UR), N-fixing species planting (PN), single-species retention (RS), and diverse vegetation retention (RD), in August 2017. The UR treatment was to remove all understory vegetation and cover the soil surface with black weed barrier fabric to prevent vegetation regrowth. The PN treatment involved seeding Sesbania cannabina after eliminating all vegetation to ensure comparable cover as natural vegetation. The RS treatment preserved E. crusgalli, the dominant understory species, by eliminating other species and seeding to ensure comparable cover as natural vegetation. The RD treatment did not involve any interference to the understory throughout the study period to maintain a natural understory with diverse species. A randomized complete block design was employed using three blocks, each containing four 42 m × 12 m plots. Treatments were randomly assigned within blocks and maintained annually according to the experimental design.

2.2. Soil Sampling and Basic Properties Determination

Mineral soil from the 0–10 cm and 10–20 cm layers was collected using stainless-steel soil cores from 3 soil profiles at randomized points in each experimental plot in August 2023, six years after understory vegetation treatments. After being transported at below 4 °C to the laboratory and sieved through a 2 mm mesh, each soil sample was divided into two aliquots; one was stored at 4 °C for the analysis of ammonium (NH4+-N), nitrate (NO3−-N), and dissolved organic nitrogen (DON) contents, as well as N transformation parameters. The other aliquot was preserved at −80 °C for the subsequent metagenomic sequence of soil microorganisms and molecular analysis of N-cycling functional genes. For the analysis of N transformation parameters and metagenomic sequences, soil samples of the same soil layer from 3 sampling points within the same plot were mixed in equal weight to form a composite sample prior to analysis.
Soil NH4+-N, NO3−-N, and DON was extracted using a 2 mol/L KCl solution at a 1:5 (w/v) soil-to-solution ratio by shaking for 1 h (180 r min−1). The NH4+-N concentration was determined by the indophenol blue method, and the NO3−-N concentration was determined by the phenol disulfonic acid method using a flow injection (AA3, Bran+Luebbe, Norderstedt, Germany). The DON concentration was calculated as the difference between total dissolved N (TDN) and inorganic N, with TDN being determined by converting all N in the extraction into nitrate form with alkaline persulfate oxidation digestion followed by measuring the NO3−-N concentration via dual-wavelength ultraviolet spectrophotometry (UV-Vis, Analytik Jena AG, Jena, Germany).

2.3. Soil Gross N Transformation

Soil gross N transformation rates were determined using the 15N isotope dilution method. Four fresh soil aliquots of equal weight (20 g) were prepared for each soil sample in 300 mL glass vessels. Two aliquots were homogeneously injected with 1 mL of 15N-(NH4)2SO4 solution (10 µg N/L with 10 atom% 15N), while the other two were injected with 1 mL of 15N-KNO3 solution (10 µg N/L with 10 atom% 15N). The vessels were covered using paraffin film and dark-incubated at 25 °C. After 15 min and 24 h after incubation, half of the replicates were retrieved and immediately extracted with 2 mol/L KCl solution via the same methods of inorganic N extraction. After analyzing the concentration of NH4+-N and NO3−-N, the 15N enrichments of NH4+-N and NO3−-N were subsequently determined by isotope mass spectrometry (DELTAV Advantage, ThermoFisher Scientific, Waltham, MA, USA) after removing the inorganic N to an acidified glass-fiber disk via the diffusion method [35]. Gross rates of N mineralization (GM), NH4+ immobilization (NIM), and nitrification (GN) were calculated separately using the model of Mary et al. [36].

2.4. Soil Macrogenomic Analysis

Total soil genomic DNA was extracted using the OMEGA Mag-Bind Soil DNA Kit and sequenced via Illumina Nova Seq shotgun metagenomics (Illumina Inc., San Diego, CA, USA) with DNA quality verified by concentration (≥2 ng/μL) and purity (A260/A280). Metagenomic libraries were sequenced on the Illumina NovaSeq 6000 platform (PE150, 400 bp insert size), yielding ~20 million reads per sample. Raw data were quality-filtered using fastp (v0.23.2; Q ≥ 20, read length ≥ 50 bp, N < 30%). Taxonomic annotation was performed using Kaiju (v1.9.0; maximum allowed mismatches set to 5, E-value threshold of 1×10-5) against the NCBI nr database (2022; ≥80% similarity), followed by reference genome alignment via Minimap2. Functional genes were annotated using NCycDB (v2.0) and MMseqs2 (≥95% similarity), with abundance quantified by mapped read counts. Nitrogen cycling genes (mineralization, nitrification, fixation) were prioritized for analysis.

2.5. Statistical Analyses

All experimental results were reported as mean values with standard errors (mean ± SE). Because the block effects showed no significant impact on any experimental results, statistical differences between the understory vegetation treatments were evaluated using one-way ANOVA without block effect analysis after normality and homoscedasticity validation, implemented in SPSS 22.0 (version 2021, https://www.ibm.com/cn-zh/spss, accessed on 15 July 2024). Fisher’s Least Significant Difference (LSD) test was employed for mean comparisons. All figures were created using Origin 2024b software (version 2024b, https://www.originlab.com, accessed on 1 August 2024). Stepwise multiple linear regression analyses were employed to elucidate the regulating effects of key functional genes on gross N mineralization rate, nitrification rate, and immobilization rate.

3. Results

3.1. Soil N Transformation Under Different Understory Vegetation Treatments

After 6 years of understory vegetation manipulation, soil labile N content showed significant differences among the treatments, in a descending order of RD > PN > RS > UR (Figure 1). UR treatment had a significantly lower DON content than RS (by 38%, p = 0.01), PN (by 60%, p < 0.001), and RD (by 94%, p < 0.001) in the 0–10 cm soil layer. The 10–20 cm layer exhibited slightly different patterns as the PN treatment retained significantly higher values than UR, while no significant difference was observed between the PN and RS treatments. Ammonia content showed consistent vertical distribution patterns, with the RD treatment yielding significantly higher content than RS (p < 0.001) and PN treatment (p < 0.001) across both soil layers. No significant differences in soil NO3−-N content were observed among the four understory vegetation treatments across both soil layers(p = 0.285, p = 0.268).
The gross rates of N mineralization (GM) and NH4+ immobilization (NIM) exhibited a decreasing trend with soil depth (Figure 2). Compared to UR, all the vegetation retention treatments enhanced GM rate by 40.0–250.0% and NIM rate by 17.0–136.0% in the 0–10 cm layer. The PN treatment exhibited a higher GM rate (30.6%, p = 0.56) and significantly higher NIM rate (102.3%, p = 0.01) than the RS treatment, while the RD treatment showed even greater enhancements of GM (149.3%, p = 0.004) and NIM (84.8%, p = 0.001) relative to RS. In the 10–20 cm layer, all vegetation retention treatments increased GM by 303.0–397.0% and NIM by 54.0–129.0% compared to UR treatment, with PN and RD showing enhancement effects. Nitrification (GN) rates in both soil layers were consistently higher under all vegetation retention treatments than vegetation removal (Figure 2b). In the 0–10 cm layer, the PN and RS treatments increased nitrification rates by 221.1% and 120.3%, respectively, while the RD treatment showed a more pronounced 363.1% enhancement in GN rate compared to the UR treatment. In the 10–20 cm layer, PN, RS, and RD treatments enhanced nitrification rates by 13.5-fold, 19.7-fold, and 81.9-fold, respectively, compared to UR treatment.

3.2. Soil Microbial Community Composition Under Different Understory Vegetation Treatments

The composition of soil microbial communities shifted significantly among different understory treatments (Figure 3). The presence of understory notably increased the relative abundance of the phylum Proteobacteria while decreasing that of phylum Actinobacteria compared to the UR treatment. Compared to the RS treatment, PN led to a significant increase in the relative abundance of the phyla Chloroflexi (by 68.2%, p = 0.01) and Nitrospirae (by 25.7%, p = 0.04). Additionally, the relative abundance of the phyla Verrucomicrobia and Planctomycetota was greater in the RD treatment compared to RS. Relative to the UR treatment, all vegetation retention treatments remarkably increased the relative abundance of the phylum Basidiomycota while decreasing that of the phylum Ascomycota. Conversely, both PN and RD treatments resulted in a reduction in the relative abundances of the phyla Basidiomycota, Mucoromycota, and Verrucomicrobia when compared to the RS treatment.

3.3. Functional Genes Related to N Transformation Under Different Understory Vegetation Treatments

Compared to understory removal, all vegetation retention treatments enhanced the abundance of functional genes associated with soil N transformation (Figure 4). Specifically, the PN treatment increased gene abundance related to N mineralization (by 2.7%), immobilization (3.8%), and nitrification (14.9%) relative to the RS treatment, while the RD further upregulated these functional genes. For the function genes related to N mineralization, the RS treatment significantly increased the abundances of gdh_K15371 (by 7%, p < 0.001) and ureB (by 8%, p = 0.07) compared to UR treatment, with PN and RD treatments showing further enhancements compared to UR treatment (Figure 5). Regarding the function genes related to nitrification, the RS treatment elevated hao gene abundance by 38.3% relative to UR treatment (p < 0.001), while the PN and RD treatments exhibited greater increases of 54.0% (p < 0.001) and 56.7% (p < 0.001), respectively. Notably, PN treatment had a significantly lower amoC_A abundance compared to RS treatment. All vegetation retention treatments decreased N immobilization gene gs_K00264 abundance by 8% compared to UR treatment. Compared to the PN and RD treatment, RS treatment significantly decreased the abundance of gs_K00266 and asnB.

3.4. Contribution Analysis of Microbial Composition, Functional Genes, and N Transformation

Metagenomic analysis revealed that the top 10 microbial contributors to N mineralization, nitrification, and N immobilization were exclusively bacterial phyla (Figure 6). The dominant phylum was Proteobacteria for mineralization, Actinobacteria for immobilization, and Nitrospirae for nitrification, respectively. Compared with the UR treatment, all vegetation retention treatments enhanced the contribution of the bacterial phylum Proteobacteria but significantly reduced that of Actinobacteria to N transformation. Compared to the RS treatment, the PN treatment markedly increased the contributions of the phyla Proteobacteria and Nitrospirae to N mineralization and nitrification, along with boosting the contribution of phylum Chloroflexi to N immobilization. The RD treatment elevated the contribution of the phyla Proteobacteria, Chloroflexi, and Nitrospirae to N mineralization by 9%, 18%, and 58%, respectively, compared to RS treatment.
Multiple stepwise regression analyses were employed to assess the relative importance of the associated functional genes in regulating GM, GN, and NIM across different understory vegetation treatments (Table 1). The GM rate was strongly controlled by gene gdh_K15371 positively, followed by ureB, while gdh_K00261 exhibited significant negative regulation. For GN, amoA_B demonstrated the strongest positive regulation, with hao showing a secondary positive influence, whereas amoC_B acted as a significant negative regulator. Notably, NIM was predominantly positively regulated by gs_K00284. These findings quantitatively establish the hierarchical control of key functional genes in soil N transformation processes under varying understory vegetation regimes, with gdh_K15371, amoA_B, and gs_K00284 emerging as pivotal genetic determinants for mineralization, nitrification, and N immobilization, respectively.

4. Discussion

After 6 years of treatment, understory vegetation removal significantly reduced the gross rate of soil N mineralization (40–250%), nitrification (44–363%), and NH4+ immobilization (16–135%), and consequently decreased soil-available N (35–121%) in the 0–10 cm soil layer compared to the three vegetation-retaining treatments, while no significant changes were observed in the 10–20 cm soil layer (Figure 1 and Figure 2). The inhibitory effect in the 0–10 cm soil layer may be attributed primarily to the decrease in microbial substrate supply due to diminished organic inputs after understory removal [37,38]. Also, exacerbated microclimate alteration, such as surface soil drought after vegetation removal and soil surface exposure, might be another reason for the decrease in N transformation [39,40]. In addition, the exacerbation of soil DON leaching caused by vegetation removal may also be an important reason for reducing soil microbial activity for N transformation in the surface soil [41]. No significant changes in the 10–20 cm soil layer indicated that the subsurface soil was less affected by understory vegetation.
Compared to the treatment involving single-species retention, planting N-fixing species in the understory significantly increased soil gross N mineralization and nitrification by 83% and 53% in the 0–10 cm soil layer (Figure 2); additionally, soil labile N increased by 14% (Figure 1). This finding aligns well with an investigation in tropical grassland, where the introduction of N-fixing plants enhanced nitrification and mineralization rates 6-fold and 3-fold, respectively [42]. This consistency may be attributed to the lower C/N ratios typically found in the litter of N-fixing plants, which facilitates microbial decomposition and promotes N mineralization, ultimately enhancing soil N availability [43]. However, the enhancement of mineralization and nitrification observed in our study was relatively modest. This discrepancy might be attributed to climatic conditions and soil characteristics; high temperatures and humidity typically found in tropical regions generally favor microbial activity for litter decomposition and N transformation, whereas the subtropical conditions of our study may impose limitations related to temperature and moisture [44].
Retention of diverse vegetation with high species richness significantly increased soil labile N contents in the 0–10 cm soil layer by 44–64% and enhanced the gross rate of N mineralization, NH4+ immobilization, and nitrification by 149%, 85%, and 222%, respectively, compared to the treatment involving single-species retention (Figure 1 and Figure 2). This finding aligns closely with a report that increased plant species diversity significantly elevated the soil gross N mineralization and nitrification rates by 3 and 4 times, respectively, in the subtropical karst forest ecosystem of southwest China [22]. This consistency likely stems from diverse plant communities supplying more complex carbon substrates via litter decomposition and root exudates, which better sustain varied microbial metabolic demands, maintaining overall community activity [45]. Furthermore, abundant carbon inputs promote microbial community succession toward higher functional activity, particularly by increasing the biomass and metabolic potential of key N-cycling taxa (e.g., ammonifiers and nitrifiers), thereby enhancing microbial-driven N mineralization efficiency [46]. However, some studies have found that increased vegetation diversity does not significantly improve soil N availability [47]. In the latter study, the primary plant species were shrubs and forest trees, which possessed substantial biomass and had extended growth cycles, resulting in a relatively high demand for and uptake of N [47]. In contrast, our study predominantly examines the understory vegetation, which primarily consists of herbaceous plants. These plants exhibit a comparatively lower capacity for N absorption, thereby facilitating the accumulation of available N in the soil.
Our results indicated that understory removal significantly decreased the relative abundance of soil bacterial phyla Proteobacteria and Basidiomycota, while simultaneously increasing that of Actinobacteria, Ascomycota, and Mucoromycota in comparison to all the understory vegetation retention treatments (Figure 3). This shift of microbial composition presumably resulted from diminished labile plant-derived carbon inputs after understory vegetation removal, which caused soil conditions to deteriorate and suppressed copiotrophic Proteobacteria which showed high dependence on oil organic carbon availability [48]. In contrast, oligotrophic-adapted taxa such as Actinobacteria, Ascomycota, and Mucoromycota exhibited competitive advantages under resource-limited conditions [49,50,51]. Zhao et al. [52] found that intensive mowing significantly increased the relative abundance of Actinobacteria in soil, which was consistent with our observation. However, some studies have reported the opposite results, indicating that the presence of understory vegetation increased the relative abundance of Actinobacteria [53]. These discrepancies may be attributed to the influence of inherent soil properties on microbial communities. Additionally, seasonal variations in temperature and moisture, as well as differences in humus layer properties induced by different understory vegetation types, could contribute to the alterations in soil microbial community structure [54]. The Basidiomycota are generally obligate aerobes that thrive in well-aerated soil conditions [55]; thus, understory vegetation removal reduced root-derived porosity, compromising soil aeration and ultimately diminishing Basidiomycota abundance.
Planting N-fixing species in the understory significantly increased the relative abundance of the soil bacterial phyla Nitrospirae and Chloroflexi when compared to the treatment involving single-species retention (Figure 3). N-fixing plants convert N2 to ammonia via rhizobial symbiosis. As key nitrifiers, Nitrospirae generally increased in abundance correlated with ammonia supply from symbiotic N fixation [56]. In our study, the elevated relative abundance of Nitrospirae indicated that N-fixing plants created favorable conditions for nitrification, thereby enhancing soil nitrification activity and significantly improving soil N availability. Our results also demonstrated that the increase in species richness of understory vegetation enhanced the relative abundance of the whole bacterial community (by 4–45%) compared to the treatment with single understory plant species (Figure 3). This finding aligned with the biodiversity–ecosystem functioning theory, suggesting that higher plant diversity supports more diverse microbial communities through the increase in resource complementarity [57,58]. For instance, Zhao et al. [53] observed that vegetation restoration significantly increased soil bacterial community abundance across different ecological restoration types, which is consistent with our results. This phenomenon can be primarily attributed to diverse understory vegetation providing more complex and heterogeneous carbon sources and nutrient substrates through root exudates and litter inputs, thereby meeting the differential nutritional requirements of various microbial groups and promoting microbial community proliferation. In our study, the RD treatment likely enhanced soil N mineralization and nitrification processes by broadly increasing soil microbial community abundance.
The abundance of soil functional genes hao and gdh_K15371 significantly increased with N-fixing species planting rather than when retaining the single species E. crusgalli in the understory. Previous studies have demonstrated that N mineralization is typically associated with the functional gene gdh_K15371 [59], while the genes amoA_B and hao play crucial roles in nitrification by regulating the enzymatic activities involved in ammonia oxidation and nitrite oxidation [60,61], thereby influencing soil-available N content [62]. Our functional contribution analysis further revealed that planting N-fixing species notably enhances both the relative abundance and functional contribution of the phylum Nitrospirae to mineralization and nitrification (Figure 5). The shift in microbial community likely served as a key mechanism driving the enrichment of ammonia-oxidizing functional genes (e.g., amoA_B and hao) in the soil system. Soil N transformation is generally directly driven by related soil enzyme activities, and the production of corresponding enzymes by soil microorganisms is regulated to some extent by the expression of functional genes and their downstream transcription processes. Therefore, it is necessary to further study the transcriptional expression of soil microbial functional genes and the formation of corresponding enzyme functions after changes in understory vegetation characteristics.
Compared with single-species retaining treatments, retention of diverse vegetation with high species richness generally enhanced both the relative abundance of soil bacterial communities and functional genes in the 0–10 cm soil layer (Figure 4 and Figure 5). This was consistent with the findings of Qin et al. [63] in mixed forest ecosystems and Xu et al. [64] in a grassland restoration system. This phenomenon might be attributed to higher vegetation diversity, providing more diverse organic nutrients and energy sources for soil microorganisms, thereby supporting more diversified microbial populations and enhancing their proliferation and functional activity [65]. However, both mixed forests and grassland restoration lack vegetation richness gradients, which has restricted the ability to reveal the threshold effects of vegetation diversity. Also, due to the generally nonlinear relationships between vegetation diversity and microbial responses, it is difficult to determine the critical conditions for the transition from quantitative to qualitative changes in biological interactions. Further research could incorporate experiments with multi-gradient vegetation configurations to systematically analyze the cascading mechanisms between plant diversity and soil microorganisms. In addition, we only conducted research on one type of poplar clone, stand density, and site condition. In the future, the research scope should be expanded to verify the conclusions of this study under more diverse conditions, in order to improve the site quality and productivity of plantations through more reasonable understory vegetation management.

5. Conclusions

Significant differences existed in soil N transformation rates, microbial communities, and functional gene abundance associated with N transformation after 6 years of distinct understory vegetation treatments. The presence of understory accelerated soil N transformation, shifted microbial communities, and promoted gene abundance related to N transformation compared to understory vegetation removal. Planting N-fixing species in the understory specifically increased the abundance of soil bacterial phyla Nitrospirae and Chloroflexi, while an increase in plant species richness in the understory comprehensively promoted the proliferation of the soil bacterial community. Genes gdh_K15371, ureB, hao, and amoA_B were the key factors regulating soil N mineralization and nitrification. Planting N-fixing species and retaining diverse plant species in the understory significantly increased the abundance of functional genes related to N transformation, thereby enhancing N mineralization and nitrification, showing this as a key mechanism for the increase in soil N availability. The results are of benefit for exploring the effect mechanisms of understory vegetation on key functional genes involved in N transformation and the microbial community. In brief, both planting N-fixing species and retaining diverse understory vegetation were effective strategies for maintaining sustainable management of poplar plantations by increasing soil N availability. Moreover, retaining diverse understory vegetation was more cost-effective and can obviously enhance biodiversity; therefore, it can be recommended as a preferred measure for understory vegetation management.

Author Contributions

Y.T. and W.J. were mainly responsible for the conceptualization, methodology used, data evaluation, data validation, and formal analysis. Investigations and data curation were conducted by all authors. The original draft of this article was prepared by Y.T. and W.J., who were also responsible for the review and editing process of this article. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China (grant number 2021YFD2201202) and the Research and Development Program of the Third Construction Co., Ltd. of China Construction First Bureau Group.

Data Availability Statement

Data is available upon request to the corresponding author.

Conflicts of Interest

Author Ruoning Zhu was employed by the company Third Construction Co., Ltd. of China Construction First Bureau Group. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Zhou, L.; Sun, Y.J.; Saeed, S.; Zhang, B.; Luo, M. The difference of soil properties between pure and mixed Chinese fir (Cunninghamia lanceolata) plantations depends on tree species. Glob. Ecol. Conserv. 2020, 22, e01009. [Google Scholar] [CrossRef]
  2. Ghorbani, M.; Sohrabi, H.; Sadati, S.E.; Babaei, F. Productivity and dynamics of pure and mixed-species plantations of Populous deltoids Bartr. ex Marsh and Alnus subcordata C. A. Mey. For. Ecol. Manag. 2018, 409, 890–898. [Google Scholar] [CrossRef]
  3. Boulmane, M.; Oubrahim, H.; Halim, M.; Bakker, M.R.; Augusto, L. The potential of Eucalyptus plantations to restore degraded soils in semi-arid Morocco (NW Africa). Ann. For. Sci. 2017, 74, 5. [Google Scholar] [CrossRef]
  4. Zhou, X.G.; Zhu, H.G.; Wen, Y.G.; Goodale, U.M.; Zhu, Y.L.; Yu, S.F.; Li, C.T.; Li, X.Q. Intensive management and declines in soil nutrients lead to serious exotic plant invasion in Eucalyptus plantations under successive short-rotation regimes. Land Degrad. Dev. 2020, 31, 297–310. [Google Scholar] [CrossRef]
  5. Wingfield, M.J.; Brockerhoff, E.G.; Wingfield, B.D.; Slippers, B. Planted forest health: The need for a global strategy. Science 2015, 349, 832–836. [Google Scholar] [CrossRef]
  6. Zhao, J.; He, X.; Wang, K. A hypothetical model that explains differing net effects of inorganic fertilization on biomass and/or abundance of soil biota. Theor. Ecol. 2015, 8, 505–512. [Google Scholar] [CrossRef]
  7. Nguyen, H.; Herbohn, J.; Lamb, D.; Lamb, D.; Clendenning, J.; Meadows, J. A synthesis of the available evidence to guide the design of mixed-species forest plantings for smallholder and community forestry. Small Scale For. 2018, 17, 105–123. [Google Scholar] [CrossRef]
  8. Miller, K.; Aegerter, B.A.; Brenna, J. Relationship between soil properties and nitrogen mineralization in undisturbed soil cores from California agroecosystems. Commun. Soil Sci. Plant Anal. 2019, 50, 77–92. [Google Scholar] [CrossRef]
  9. Capek, P.; Choma, M.; Tahovská, K.; Kana, J.; Kopacek, J.; Santruckova, H. Coupling the resource stoichiometry and microbial biomass turnover to predict nutrient mineralization and immobilization in soil. Geoderma 2021, 385, 114884. [Google Scholar] [CrossRef]
  10. Philippot, L.; Spor, A.; Hénault, C.; Bru, D.; Bizouard, F.; Jones, C.M.; Sarr, A. Loss in microbial diversity affects nitrogen cycling in soil. ISME J. 2013, 7, 1609–1619. [Google Scholar] [CrossRef]
  11. Ding, J.; Zhang, Y.; Deng, Y.; Cong, J.; Lu, H.; Sun, X.; Yang, C.; Yuan, T.; Li, D.; Zhou, J.; et al. Integrated metagenomics and network analysis of soil microbial community of the forest timberline. Sci. Rep. 2015, 5, 7994. [Google Scholar] [CrossRef] [PubMed]
  12. Yan, L.; Kuang, Y.; Xie, X.; Peng, K.; Deng, Y.; Gan, Y.; Li, Q.; Zhang, Y. Insights into nitrogen biogeochemical cycling in mangrove wetland from genome-resolved metagenomic sequencing. J. Hydrol. 2024, 640, 131741. [Google Scholar] [CrossRef]
  13. Lindsay, E.A.; Gibb, N.L.; Wakelin, S.A. The abundance of microbial functional genes in grassy woodlands is influenced more by soil nutrient enrichment than by recent weed invasion or livestock exclusion. Appl. Environ. Microbiol. 2010, 76, 5547–5555. [Google Scholar] [CrossRef]
  14. Yan, J.F.; Wang, L.; Hu, Y.; Tsang, Y.F.; Zhang, Y.N.; Wu, J.H.; Fu, X.H.; Sun, Y. Plant litter composition selects different soil microbial structures and in turn drives different litter decomposition pattern and soil carbon sequestration capability. Geoderma 2018, 319, 194–203. [Google Scholar] [CrossRef]
  15. Prescott, C.E.; Vesterdal, L. Decomposition and transformations along the continuum from litter to soil organic matter in forest soils. For. Ecol. Manag. 2021, 498, 119522. [Google Scholar] [CrossRef]
  16. Zhu, H.; Gong, L.; Luo, Y.; Tang, J.; Ding, Z.; Li, X. Effects of litter and root manipulations on soil bacterial and fungal community structure and function in a Schrenk’s spruce (Picea schrenkiana) forest. Front. Plant Sci. 2022, 13, 849483. [Google Scholar] [CrossRef]
  17. Wang, J.; Zhang, A.; Zheng, Y.; Song, J.; Ru, J.; Zheng, M.; Hui, D.; Wan, S. Long-term litter removal rather than litter addition enhances ecosystem carbon sequestration in a temperate steppe. Funct. Ecol. 2021, 35, 2799–2807. [Google Scholar] [CrossRef]
  18. Min, K.; Zheng, T.; Zhu, X.; Bao, X.; Lynch, L.; Liang, C. Bacterial community structure and assembly dynamics hinge on plant litter quality. FEMS Microbiol. Ecol. 2023, 99, fiad118. [Google Scholar] [CrossRef]
  19. Forrester, D.I.; Bauhus, J.; Cowie, A.L.; Vanclay, J.K. Mixed-species plantations of Eucalyptus with nitrogen-fixing trees: A review. For. Ecol. Manag. 2006, 233, 211–230. [Google Scholar] [CrossRef]
  20. Gou, X.; Reich, P.B.; Qiu, L.; Shao, M.; Wei, G.; Wang, J.; Wei, X. Leguminous plants significantly increase soil nitrogen cycling across global climates and ecosystem types. Glob. Change Biol. 2023, 29, 4028–4043. [Google Scholar] [CrossRef]
  21. Reed, S.C.; Cleveland, C.C.; Townsend, A.R.; Futuyma, D.J.; Shaffer, H.B.; Simberloff, D. Functional ecology of free-living nitrogen fixation: A contemporary perspective. Annu. Rev. Ecol. Evol. Syst. 2011, 42, 489–512. [Google Scholar] [CrossRef]
  22. Zhu, Z.; Du, H.; Gao, K.; Fang, Y.; Wang, K.; Zhu, T.; Zhu, J.; Cheng, Y.; Li, D. Plant species diversity enhances soil gross nitrogen transformations in a subtropical forest, southwest China. J. Appl. Ecol. 2023, 60, 1364–1375. [Google Scholar] [CrossRef]
  23. Ding, K.; Zhang, Y.; Liu, H.; Yang, X.; Zhang, J.; Tong, Z. Soil bacterial community structure and functions but not assembly processes are affected by the conversion from monospecific Cunninghamia lanceolata plantations to mixed plantations. Appl. Soil Ecol. 2023, 185, 104775. [Google Scholar] [CrossRef]
  24. Pereira, A.P.; Durrer, A.; Gumiere, T.; Gonçalves, J.L.; Robin, A.; Bouillet, J.P.; Wang, J.; Verma, J.P.; Singh, B.K.; Cardoso, E.J. Mixed Eucalyptus plantations induce changes in microbial communities and increase biological functions in the soil and litter layers. For. Ecol. Manage. 2019, 433, 332–342. [Google Scholar] [CrossRef]
  25. Maxwell, T.L.; Fanin, N.; Parker, W.C.; Bakker, M.R.; Belleau, A.; Meredieu, C.; Munson, A.D. Tree species identity drives nutrient use efficiency in young mixed-species plantations, at both high and low water availability. Funct. Ecol. 2022, 36, 2069–2083. [Google Scholar] [CrossRef]
  26. Liu, D.; Liu, Y.; Fang, S.; Tian, Y. Tree species composition influenced microbial diversity and nitrogen availability in rhizosphere soil. Plant Soil Environ. 2015, 61, 438–443. [Google Scholar] [CrossRef]
  27. Pereira, A.P.; Araujo, A.S.; Santana, M.C.; Lima, A.Y.; Araujo, V.L.; Verma, J.P.; Cardoso, E.J. Enzymatic stoichiometry in tropical soil under pure and mixed plantations of Eucalyptus with N2-fixing trees. Sci. Agric. 2023, 80, e20210283. [Google Scholar] [CrossRef]
  28. Xanthopoulos, G.; Radoglou, K.; Derrien, D.; Spyroglou, G.; Angeli, N.; Tsioni, G.; Fotelli, M.N. Carbon sequestration and soil nitrogen enrichment in Robinia pseudoacacia L. post-mining restoration plantations. Front. For. Global Change 2023, 6, 1190026. [Google Scholar] [CrossRef]
  29. Zhang, J.J.; Li, Y.; Chang, S.X.; Jiang, P.; Zhou, G.; Liu, J. Understory vegetation management affected greenhouse gas emissions and labile organic carbon pools in an intensively managed Chinese chestnut plantation. Plant Soil 2014, 376, 363–375. [Google Scholar] [CrossRef]
  30. Zhang, J.Y.; Qin, G.Z.; Zhai, Z.; Zhou, S.C.; Tang, L.Z.; Tian, Y. Diverse understory vegetation alleviates nitrogen competition with crop trees in poplar plantations. Forests 2021, 12, 705. [Google Scholar] [CrossRef]
  31. Deng, J.J.; Fang, S.; Fang, X.M.; Kuang, Y.W.; Lin, F.; Liu, C. Forest understory vegetation study: Current status and future trends. For. Res. 2023, 3, 6. [Google Scholar] [CrossRef] [PubMed]
  32. Fang, S.Z. Silviculture of poplar plantation in China: A review. Chin. J. Appl. Ecol. 2008, 19, 2308–2316. [Google Scholar] [CrossRef]
  33. Ge, X.M.; Tian, Y.; Tang, L.Z. Nutrient distribution indicated whole-tree harvesting as a possible factor restricting the sustainable productivity of a poplar plantation system in China. PLoS ONE 2015, 10, e0125303. [Google Scholar] [CrossRef]
  34. Yan, Y.; Fang, S.; Tian, Y.; Song, H.; Dun, X. The response of understory plant diversity and nutrient accumulation to stand structure of poplar plantation. Chin. J. Ecol. 2014, 33, 1170–1177. [Google Scholar] [CrossRef]
  35. Holmes, R.M.; Mcclelland, J.W.; Sigman, D.M.; Fry, B.; Peterson, B.J. Measuring 15N–NH4+ in marine, estuarine and fresh waters: An adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Mar. Chem. 1998, 60, 235–243. [Google Scholar] [CrossRef]
  36. Mary, B.; Recous, S.; Robin, D. A model for calculating nitrogen fluxes in soil using 15N tracing. Soil Biol. Biochem. 1998, 30, 1963–1979. [Google Scholar] [CrossRef]
  37. Matsushima, M.; Chang, S.X. Effects of understory removal, N fertilization, and litter layer removal on soil N cycling in a 13-year-old white spruce plantation infested with Canada bluejoint grass. Plant Soil 2007, 292, 243–258. [Google Scholar] [CrossRef]
  38. Trentini, C.P.; Villagra, M.; Pámies, D.G.; Laborde, V.B.; Bedano, J.C.; Campanello, P.I. Effect of nitrogen addition and litter removal on understory vegetation, soil mesofauna, and litter decomposition in loblolly pine plantations in subtropical Argentina. For. Ecol. Manag. 2018, 429, 133–142. [Google Scholar] [CrossRef]
  39. Kopecky, M.; Hederová, L.; Macek, M.; Klinerová, T.; Wild, J. Forest plant indicator values for moisture reflect atmospheric vapour pressure deficit rather than soil water content. New Phytol. 2024, 244, 1801–1811. [Google Scholar] [CrossRef]
  40. Greiser, C.; Hederová, L.; Vico, G.; Wild, J.; Macek, M.; Kopecky, M. Higher soil moisture increases microclimate temperature buffering in temperate broadleaf forests. Sci. Total Environ. Agric. For. Meteorol. 2023, 345, 109828. [Google Scholar] [CrossRef]
  41. Ullah, S.; Liu, W.; Shah, J.A.; Shen, F.; Liao, Y.; Duan, H. Effects of canopy nitrogen addition and understory vegetation removal on nitrogen transformations in a subtropical forest. Forests 2024, 15, 962. [Google Scholar] [CrossRef]
  42. Gei, M.G.; Powers, J.S. Do legumes and non-legumes tree species affect soil properties in unmanaged forests and plantations in Costa Rican dry forests? Soil Biol. Biochem. 2013, 57, 264–272. [Google Scholar] [CrossRef]
  43. Sun, L.J.; Ataka, M.; Kominami, Y.; Yoshimura, K.; Kitayama, K. A constant microbial C/N ratio mediates the microbial nitrogen mineralization induced by root exudation among four co-existing canopy species. Rhizosphere 2021, 17, 100317. [Google Scholar] [CrossRef]
  44. Yang, S.; Zhang, Y.; Cong, J.; Wang, M.; Zhao, M.; Lu, H.; Xie, C.; Yang, C.; Yuan, T.; Li, D.; et al. Variations of soil microbial community structures beneath broadleaved forest trees in temperate and subtropical climate zones. Front. Microbiol. 2017, 8, 200. [Google Scholar] [CrossRef]
  45. Sánchez, L.F.; García, M.J.; Chacón, N. Nitrogen mineralization in soils under grasses and under trees in a protected Venezuelan savanna. Acta Oecol. 1997, 18, 27–37. [Google Scholar] [CrossRef]
  46. Kundu, K.; Bergmann, I.; Hahnke, S. Carbon source—A strong determinant of microbial community structure and performance of an anaerobic reactor. J. Biotechnol. 2013, 168, 616–624. [Google Scholar] [CrossRef]
  47. Zhu, X.; Fang, X.; Xiang, W.; Chen, L.; Ouyang, S.; Lei, P. Vegetation restoration drives dynamics of soil nitrogen content and availability in the subtropics. Catena 2023, 220, 106720. [Google Scholar] [CrossRef]
  48. Fierer, N.; Bradford, M.A.; Jackson, R.B. Toward an ecological classification of soil bacteria. Ecol. Lett. 2007, 88, 1354–1364. [Google Scholar] [CrossRef]
  49. Fierer, N.; Leff, J.W.; Adams, B.J.; Nielsen, U.N.; Bates, S.T.; Lauber, C.L.; Owens, S.; Gilbert, J.A.; Wall, D.H.; Caporaso, J.G. Cross-biome metagenomic analyses of soil microbial communities and their functional attributes. Proc. Natl. Acad. Sci. USA 2012, 109, 21390–21395. [Google Scholar] [CrossRef]
  50. Lundell, T.K.; Mäkelä, M.R.; Hildén, K. Lignin-modifying enzymes in filamentous basidiomycetes—Ecological, functional and phylogenetic review. J. Basic Microbiol. 2010, 50, 5–20. [Google Scholar] [CrossRef]
  51. Cotrufo, M.F.; Wallenstein, M.D.; Boot, C.M.; Denef, K.; Paul, E. The Microbial Efficiency-Matrix Stabilization (MEMS) framework integrates plant litter decomposition with soil organic matter stabilization: Do labile plant inputs form stable soil organic matter? Glob. Change Biol. 2013, 19, 988–995. [Google Scholar] [CrossRef]
  52. Zhao, S.; Hou, X.; Wu, X.; Ding, M.; Duo, L. Lawn vegetation regulation changes the structure and function of soil bacterial community at airport. Chin. J. Ecol. 2023, 43, 5072–5083. [Google Scholar] [CrossRef]
  53. Zhao, H.; Li, X.; Zhang, Z.; Yang, J.; Zhao, Y.; Yang, Z.; Hu, Q. Effects of natural vegetative restoration on soil fungal and bacterial communities in bare patches of the southern Taihang Mountains. Ecol. Evol. 2019, 9, 10432–10441. [Google Scholar] [CrossRef] [PubMed]
  54. Liu, Y.; Wang, F.; Wang, Z.; Xiang, L.; Fu, Y.; Zhao, Z.; Kengara, F.O.; Mei, Z.; He, C.; Bian, Y.; et al. Soil properties and organochlorine compounds co-shape the microbial community structure: A case study of an obsolete site. Environ. Res. 2024, 240, 117589. [Google Scholar] [CrossRef] [PubMed]
  55. Fraatz, M.A.; Naeve, S.; Hausherr, V.; Zorn, H.; Blank, L.M. A minimal growth medium for the basidiomycete Pleurotus sapidus for metabolic flux analysis. Fungal Biol. Biotechnol. 2014, 1, 9. [Google Scholar] [CrossRef] [PubMed]
  56. Leff, J.W.; Jones, S.E.; Prober, S.M.; Barberán, A.; Borer, E.T.; Firn, J.L.; Harpole, W.S.; Hobbie, S.E.; Hofmockel, K.S.; Knops, J.M.; et al. Consistent responses of soil microbial communities to elevated nutrient inputs in grasslands across the globe. Proc. Natl. Acad. Sci. USA 2015, 112, 10967–10972. [Google Scholar] [CrossRef]
  57. Zhu, D.; Wang, P.; Zhang, W.; Yuan, Y.; Li, B.; Wang, J. Sampling and complementarity effects of plant diversity on resource use increases the invasion resistance of communities. PLoS ONE 2015, 11, 0150128. [Google Scholar] [CrossRef]
  58. Zhou, T.; Liang, G.P.; Reich, P.B.; Delgado, B.M.; Wang, C.K.; Zhou, Z.H. Promoting effect of plant diversity on soil microbial functionality is amplified over time. One Earth 2024, 7, 2139–2148. [Google Scholar] [CrossRef]
  59. Nie, S.Q.; Zhang, Z.F.; Mo, S.M.; Li, J.H.; He, S.; Kashif, M.; Liang, Z.W.; Shen, P.H.; Yan, B.; Jiang, C.J. Desulfobacterales stimulates nitrate reduction in the mangrove ecosystem of a subtropical gulf. Sci. Total Environ. 2021, 769, 144562. [Google Scholar] [CrossRef]
  60. Spasov, E.; Tsuji, J.M.; Hug, L.A.; Doxey, A.C.; Sauder, L.A.; Parker, W.J.; Neufeld, J.D. High functional diversity among Nitrospira populations that dominate rotating biological contactor microbial communities in a municipal wastewater treatment plant. ISME J. 2019, 14, 1857–1872. [Google Scholar] [CrossRef]
  61. Zhao, J.; Huang, L.B.; Chakrabarti, S.; Cooper, J.; Choi, E.; Ganan, C.; Tolchinsky, B.; Triplett, E.W.; Daroub, S.H.; Martens, H.W. Nitrogen and phosphorous acquisition strategies drive coexistence patterns among archaeal lineages in soil. ISME J. 2023, 17, 1839–1850. [Google Scholar] [CrossRef] [PubMed]
  62. Li, K.; Lin, H.; Han, M.; Yang, L. Soil metagenomics reveals the effect of nitrogen on soil microbial communities and nitrogen-cycle functional genes in the rhizosphere of Panax ginseng. Front. Plant Sci. 2024, 15, 1411073. [Google Scholar] [CrossRef] [PubMed]
  63. Qin, F.; Yang, F.; Ming, A.; Jia, H.; Zhou, B.; Xiong, J.; Lu, J. Mixture enhances microbial network complexity of soil carbon, nitrogen and phosphorus cycling in Eucalyptus plantations. For. Ecol. Manag. 2024, 553, 121632. [Google Scholar] [CrossRef]
  64. Xu, H.; Chen, C.; Chen, W.; Pang, Z.; Zhang, G.; Zhang, W.; Kan, H. Metagenomics reveals soil nitrogen cycling after vegetation restoration: Influence of different vegetation restoration strategies. Appl. Soil Ecol. 2024, 204, 105695. [Google Scholar] [CrossRef]
  65. Hättenschwiler, S.; Tiunov, A.; Scheu, S. Biodiversity and litter decomposition in terrestrial ecosystems. Annu. Rev. Ecol. Evol. Syst. 2005, 36, 191–218. [Google Scholar] [CrossRef]
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Figure 1. Soil labile nitrogen content of the (a) 0–10 cm and (b) 10–20 cm soil layers under different understory vegetation treatments (n = 36). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
Figure 1. Soil labile nitrogen content of the (a) 0–10 cm and (b) 10–20 cm soil layers under different understory vegetation treatments (n = 36). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
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Figure 2. Relations of gross nitrogen mineralization rate with (a) NH4+ immobilization rate and (b) gross nitrification rate under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Closed and open circles represent the 0–10 cm and 10–20 cm soil layers, respectively.
Figure 2. Relations of gross nitrogen mineralization rate with (a) NH4+ immobilization rate and (b) gross nitrification rate under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Closed and open circles represent the 0–10 cm and 10–20 cm soil layers, respectively.
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Figure 3. Relative abundance of the main soil microbial phyla (a) Proteobacteria, (b) Actinobacteria, (c) Chloroflexi, (d) Acidobacteria, (e) Candidatus Rokubacteria, (f) Verrucomicrobia, (g) Planctomycetota, (h) Nitrospirae, (i) Gemmatimonadetes, (j) Cyanobacteria, (k) Basidiomycota, (l) Ascomycota, (m) Mucoromycota, and (n) Chytridiomycota under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
Figure 3. Relative abundance of the main soil microbial phyla (a) Proteobacteria, (b) Actinobacteria, (c) Chloroflexi, (d) Acidobacteria, (e) Candidatus Rokubacteria, (f) Verrucomicrobia, (g) Planctomycetota, (h) Nitrospirae, (i) Gemmatimonadetes, (j) Cyanobacteria, (k) Basidiomycota, (l) Ascomycota, (m) Mucoromycota, and (n) Chytridiomycota under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
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Figure 4. Total gene abundance related to (a) nitrogen mineralization, (b) nitrification, and (c) nitrogen immobilization under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
Figure 4. Total gene abundance related to (a) nitrogen mineralization, (b) nitrification, and (c) nitrogen immobilization under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
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Figure 5. Abundance of functional genes related to (a) nitrogen mineralization, (b) nitrification, and (c) nitrogen immobilization under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
Figure 5. Abundance of functional genes related to (a) nitrogen mineralization, (b) nitrification, and (c) nitrogen immobilization under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters above the bars indicate significant difference between treatments at p < 0.05. Error bar represents standard error.
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Figure 6. Contribution of microbial phyla to (a) gross nitrogen mineralization, (b) nitrification, and (c) nitrogen immobilization under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters indicate significant difference between treatments at p < 0.05.
Figure 6. Contribution of microbial phyla to (a) gross nitrogen mineralization, (b) nitrification, and (c) nitrogen immobilization under different understory vegetation treatments (n = 12). UR, understory removal; PN, N-fixing species planting; RS, single-species retention; RD, diverse vegetation retention. Different letters indicate significant difference between treatments at p < 0.05.
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Table 1. Multiple regression analysis (stepwise) of gross N transformation with soil functional genes (n = 12).
Table 1. Multiple regression analysis (stepwise) of gross N transformation with soil functional genes (n = 12).
Variable YxStandardized CoefficientCoefficient Standard ErrorAdjusted R2pVariance Inflation Factor
Gross N mineralization rate (mg·kg−1·d−1)gdh_K153710.850.280.90<0.0012.74
gdh_K00261−0.570.14
ureB0.250.28
Gross nitrification rate (mg·kg−1·d−1)amoA_B 0.550.050.98<0.0011.26
hao0.490.05
amoC_B−0.370.04
NH4+ immobilization rate (mg·kg−1·d−1)gs_K002840.830.180.65<0.0012.20
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Jia, W.; Li, T.; Ye, P.; Chen, Y.; Zhu, R.; Yan, R.; Yue, H.; Tian, Y. Metagenomic Insights into How Understory Vegetation Enhances Soil Nitrogen Availability via Microbial Nitrogen Transformation in Poplar Plantations. Agronomy 2025, 15, 1537. https://doi.org/10.3390/agronomy15071537

AMA Style

Jia W, Li T, Ye P, Chen Y, Zhu R, Yan R, Yue H, Tian Y. Metagenomic Insights into How Understory Vegetation Enhances Soil Nitrogen Availability via Microbial Nitrogen Transformation in Poplar Plantations. Agronomy. 2025; 15(7):1537. https://doi.org/10.3390/agronomy15071537

Chicago/Turabian Style

Jia, Wenyu, Tong Li, Peilei Ye, Yuxin Chen, Ruoning Zhu, Ruixin Yan, Haoran Yue, and Ye Tian. 2025. "Metagenomic Insights into How Understory Vegetation Enhances Soil Nitrogen Availability via Microbial Nitrogen Transformation in Poplar Plantations" Agronomy 15, no. 7: 1537. https://doi.org/10.3390/agronomy15071537

APA Style

Jia, W., Li, T., Ye, P., Chen, Y., Zhu, R., Yan, R., Yue, H., & Tian, Y. (2025). Metagenomic Insights into How Understory Vegetation Enhances Soil Nitrogen Availability via Microbial Nitrogen Transformation in Poplar Plantations. Agronomy, 15(7), 1537. https://doi.org/10.3390/agronomy15071537

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