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Article

Silver Nanoparticles Alter the Diazotrophic Community Structure and Co-Occurrence Patterns in Maize Rhizosphere

by
Hui Chen
1,
Siyao Li
2,
Chengheng Fan
2 and
Jiling Cao
2,*
1
State Key Laboratory of Humid Subtropical Mountain Ecology of Ministry of Science and Technology of Fujian Province, Fujian Normal University, Fuzhou 350007, China
2
College of Land Resource and Environment, Jiangxi Agricultural University, Nanchang 330045, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(7), 1601; https://doi.org/10.3390/agronomy15071601
Submission received: 25 May 2025 / Revised: 28 June 2025 / Accepted: 29 June 2025 / Published: 30 June 2025
(This article belongs to the Section Agricultural Biosystem and Biological Engineering)
Browse Figures
Versions Notes

Abstract

Biological nitrogen (N) fixation is an ecological method used to provide nutrition for crops and reduce fertilizer application in terrestrial ecosystems. Silver nanoparticles (AgNPs) are becoming environmental contaminants, and, thus, could negatively affect the activity and diversity of soil diazotrophs. To test this, a greenhouse pot experiment for growing maize was performed under different concentrations of AgNPs (0, 1, 5, 10, 20 mg kg−1). We measured the N2-fixation activity and abundance of nifH gene encoding the nitrogenase reductase subunit and analyzed the diversity, composition and co-occurrence networks of diazotrophic communities in maize rhizospheric soil. Results showed that a lower dose of AgNPs did not show significant influence on soil diazotrophs, while a higher dose of AgNPs decreased both soil N2-fixation activity and nifH gene abundance, though diazotrophic diversity remained unchanged. AgNPs at 10 mg kg−1 and 20 mg kg−1 strongly shifted the community composition of diazotrophs, increasing the proportions of Bradyrhizobium and Paenibacillus, while decreasing Azospirillum and Rhizobium. Network analysis revealed weakened negative associations among species under AgNPs, with keystone taxa shifting from Bradyrhizobium, Geobacter, Azospirillum and Burkholderia to Bradyrhizobium, Paenibacillus and Skermanella under AgNPs. Soil-soluble Ag, dissolved organic carbon and soil pH were identified as the factors most closely driving the diazotrophic community composition. In conclusion, higher doses of AgNPs could inhibit N2-fixation activity and shape the diazotrophic communities. These findings provide empirical evidence of AgNPs’ ecological impacts on soil microbial functions.

1. Introduction

Silver nanoparticles (AgNPs) have been considered as potential environmental contaminants due to the wide usage of AgNPs as antimicrobial additives in industrial, commercial and agricultural applications [1,2]. AgNPs could enter agricultural soils through irrigation of wastewater and application of nano-containing pesticides and fertilizers, and, thus, show risks to agroecosystems [3]. AgNPs have impacts on the growth and biomass of aboveground plants, but also the activity and composition of belowground microbial communities [4,5]. Diverse functional communities of microbes interact with plants and serve as key drivers in biological cycling; thus, it is crucial to explore the impact of AgNPs on microbial communities of functional traits to link microbes to soil biochemical cycling and plant growth.
Soil diazotrophs play an important role in the nitrogen cycle by converting atmospheric N2 into bioavailable forms [6]. In agricultural ecosystems, microbiological N2 fixation could contribute an estimated 32 Tg nitrogen (N) per year [7]. Therefore, biological nitrogen fixation is an ecological method to provide nitrogen nutrition to maintain crop growth and productivity. Many environmental factors, such as soil pH [8], soil carbon/nitrogen contents [9] and heavy metals [10], influence the diazotrophic community. A recent study found that copper oxide NPs increased the soil N2-fixation activity and shifted the diazotrophic community composition [11]. AgNPs may also alter diazotrophic abundances and community structure via selecting specific diazotrophic taxa. Moreover, AgNPs are also expected to indirectly affect diazotrophic communities via plant–microbe interactions based on their phytotoxicity. It is still unclear how soil diazotrophs change with AgNP doses, which is important for agriculture ecosystem management.
Maize (Zea mays L.), a globally important agronomic crop [12], has shown various responses to AgNPs, including promotion of seed germination [13], inhibited plant growth [14], and shifted bacterial community composition [4]. However, its effects on soil diazotrophs are poorly understood. This study aimed to address how AgNP levels affect N2-fixation activity and the diazotrophic community in maize rhizospheric soil, and to identify the underlying mechanism of AgNPs on diazotrophs. It was hypothesized that AgNPs would inhibit the N2-fixation activity and decrease the diazotrophic diversity due to their excellent antimicrobial properties. Furthermore, it was hypothesized that AgNPs would alter the community composition and co-occurrence network due to the differential tolerance abilities among diazotrophic taxa.

2. Materials and Methods

2.1. Experimental Design

Soil used in this study was collected from an agricultural field in the Agro-Ecological Experimental Station of Fengqiu County (35°00′ N, 114°24′ E), Henan Province, China, then sieved to 2 mm and stored for pot experiments. Soil in this region has a sandy loam texture with 9% clay and 21.8% silt. It was categorized as Aquic Inceptisol (a calcareous fluvo-aquic soil). Soil properties had a pH of 8.60 and contained 0.58% of organic matter, 0.045% of total N, 0.050% of total P and 1.86% of total K. AgNPs were purchased from the Aladdin Company (Shanghai, China) and were composed of silver with a spherical shape (40 nm).
Given the increasing release of AgNPs into the environment [1,2], a wide range of AgNPs (1, 5, 10, 20 mg kg−1 air-dried soil) was used to study their biological effects on soil ecosystems. No Ag addition was used as the control. In total, five treatments were established. Each treatment had five replicates. Each pot (10.8 cm in height, 12 cm in top diameter) contained AgNPs (at 0.5 mg [1 mg kg−1], 2.5 mg [5 mg kg−1], 5 mg [10 mg kg−1] or 10 mg [20 mg kg−1]) and 500 g of soil to form the AgNP-amended mixture. Maize seeds were germinated (28 °C) for two days. Two healthy seeds of uniform size were transplanted to plastic pots containing AgNP-amended soils. In May 2024, maize seedlings were grown in natural sunlight conditions in a greenhouse. All maize plants were irrigated with Hoagland solution every two weeks after transplanting for ten days.

2.2. Plant and Soil Sample Collection

After 30 days of planting, a time at which the impacts of AgNPs on plant performance are readable, aboveground and belowground plant tissues were separately collected to measure the dry plant mass. Briefly, aboveground plant tissues were firstly cut from root systems. Belowground roots were harvested by breaking apart the soil. All aboveground and belowground tissues of maize plants were thoroughly cleaned with tap water and deionized water and weighed after oven-drying at 70 °C for 2 days. Rhizosphere soil samples were collected, homogenized and sieved through a 2 mm mesh. The sieved soils were then subsampled, with one part stored at −80 °C for DNA extraction and the other stored at 4 °C for chemical measurement.

2.3. Soil Chemical Properties and N2-Fixation Activity Determination

Soil pH and EC were determined in deionized water (soil/water = 1:2.5) with a pH meter (FE20-FiveEasy™ pH, Mettler Toledo, Zurich, Switzerland) and an EC meter (Orion Lab Star EC112, Thermo Scientific, MA, USA). Soil total nitrogen (TN) was measured by Kjeldahl digestion [15]. Soil dissolved organic carbon (DOC) was determined by a TOC analyzer (Shimadzu, Kyoto, Japan) after K2SO4 extraction. Soil available P (AP) and available K (AK) were measured with the molybdenum-blue method and flame photometry [16]. Soil available Ag was determined by a ICP-MS analyzer (Thermo Scientific, Waltham, MA, USA) after extracting with diethylene triamine pentaacetic acid (DTPA) [17]. Given that nitrogenase can reduce acetylene to ethylene, soil N2-fixation activity was determined with the acetylene reduction method [18].

2.4. Soil nifH Gene Quantification and High-Throughput Sequencing

Soil microbial DNA was extracted from 0.5 g of samples using a Fast DNA® SPIN Kit (MP Biomedicals, Santa Ana, CA, USA). The concentration and quality of extracted DNA were analyzed by ND-2000c UV-vis spectrophotometry (Thermo Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis. Thereafter, DNA was stored at −20 °C for further usage.
The copy number of the nifH gene was quantified by real-time quantitative PCR (qPCR) with a CFX96Tm Real-time System (Bio-Rad, Hercules, CA, USA). Amplification was conducted with the PolyF/PolyR primer pairs in a 25 μL reaction mixture containing 12.5 μL of SYBR Premix Ex Taq (TaKaRa, Osaka, Japan), 0.5 μL of forward and reverse primer, 2 μL DNA template and 9.5 μL of sterile H2O [19]. The qPCR program was carried out as described by [11]. Three replicates were performed for each sample. The plasmid pMS 18-T (TaKaRa, Osaka, Japan) containing the nifH gene fragment was used to generate standard curves for qPCR. The qPCR products were specified by melt curves and electrophoresis on a 1.8% agarose gel. PCR amplification efficiencies were 97–104% with R2 of 0.98–0.99. Data were analyzed using the Cycler (Bio-Rad, Hercules, CA, USA).
The nifH gene was amplified with the same primer set as those used in qPCR. Each reaction mixture (50 μL) contained 4 μL of deoxynucleoside triphosphates (2.5 mM), 2 μL of each primer (10 μM), 0.4 μL of Taq DNA polymerase (2 U) and 1 μL of template DNA (50 ng). Amplification was started with an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 10 s, 55 °C for 20 s, 72 °C for 30 s and finally an extension at 72 °C for 7 min. PCR was run in triplicate. The PCR products of three replicates were pooled and purified with a PCR purification kit (Qiagen, Hilden, Germany). After that, the purified PCR products were normalized and mixed in equimolar amounts and then sequenced using the Illumina MiSeq system (Illumina, San Diego, CA, USA).

2.5. Bioinformatic Analysis

All sequencing data were processed with the Quantitative Insights into Microbial Ecology (QIIME, 1.9.1) platform [20]. In brief, low-quality sequences (score < 20 or length < 400 bp) were removed. The residual sequences were translated into amino acid sequences using the FunGene Pipeline references. Sequences that failed to match the nifH protein were deleted. Thereafter, the remaining high-quality sequences were clustered into operational taxonomic units (OTUs) at a 95% similarity level with UCLUST. Finally, OTUs were taxonomically assigned by searching representative sequences against the nifH reference database. To remove the sampling effects, all samples were rarefied to 84,752 sequences to analyze the diazotrophic community. Alpha diversity was evaluated by calculating observed OTUs, Chao1, ACE and Good’s coverage indices.

2.6. Statistical Analysis

One-way analysis of variance (ANOVA) was performed to analyze the significant differences (p < 0.05) of soil diazotrophic diversity, abundances and N2-fixation activity among different treatments with SPSS 18.0 (SPSS Inc., Chicago, IL, USA). Non-metric multidimensional scaling (NMDS) and principal coordinate analysis (PCoA) based on Bray–Curtis distance were used to visualize diazotrophic community composition. Response ratio method was conducted to display the significant changes in diazotrophic abundances to AgNPs relative to the control [21]. Phylogenetic molecular ecological networks (pMENs) were performed with the random matrix theory (RMT) to explore the interactions between diazotrophic taxa [22]. Network parameters were analyzed to quantify the topological properties of networks under different treatments. The hub nodes were sorted into four categories (peripherals, connectors, module hubs, and network hubs) based on Zi and Pi [23]. Microbial ecological networks were visualized by the Gephi platform 0.9.1. A Mantel test was conducted to analyze the linkages of soil diazotrophic community composition with environmental factors. The correlations between diazotrophic abundances and soil properties and plant biomass were analyzed by linear regression analyses.

3. Results

3.1. Soil Properties

Compared to the control (0 mg kg−1), AgNPs at 20 mg kg−1 significantly decreased soil pH, AP and DOC contents (p < 0.05). AgNPs at 5 mg kg−1 and 10 mg kg−1 also tended to reduce soil pH. Soil-soluble Ag content increased along with an increase in AgNP concentrations. In contrast, no significant changes in soil EC, TN and AK contents were detected across different concentrations of AgNPs relative to the control (Table 1).

3.2. Soil N2-Fixation Activity and nifH Gene Abundance

AgNPs showed adverse effects on the soil N2-fixation activity and nifH gene abundance (Figure 1a,b). Compared to the control, all studied AgNPs (1, 5, 10, 20 mg kg−1) significantly decreased (p < 0.05) the N2-fixation activity, with the highest reduction found in 20 mg kg−1 AgNPs. AgNPs at 10 mg kg−1 and 20 mg kg−1 significantly decreased the nifH gene abundance, especially 20 mg kg−1 AgNPs.

3.3. Soil Diazotrophic Community Composition and Diversity Indices

A total of 3,037,790 sequences were obtained from 15 soil samples by high-throughput sequencing (Table S1). The Good’s coverage values were higher than 0.995 (Table 2), indicating that most of the diazotrophic taxa were captured to study the soil diazotrophic community characteristics. Regardless of the richness and diversity of the diazotrophic community, AgNPs did not significantly alter Chao1, Observed OTUs, ACE, Shannon and Simpson (Table 2).
Based on 95% similarity, high-quality sequences were clustered into 1207 OTUs, with a majority being assigned to Proteobacteria (~81.9%). Firmicutes and Spirochaetes accounted for approximately 13.8% and 0.99% (Figure S1a). Proteobacteria was the most dominant phyla in all experimental treatments. No significant changes in the relative abundances of diazotrophs at the phyla level were observed under AgNPs (Figure S1b). At the order level, Rhizobiales (~39.8%), Rhodospirillales (~31.2%) and Desulfuromonadales (~5.76%) were the three dominant members. The relative abundance of Rhizobiales increased and then decreased along with the increases in AgNP concentrations. In contrast, the relative abundances of Rhodospirillales decreased and then increased under AgNPs. AgNPs increased the relative abundance of Desulfuromonadales (Figure S2). At a higher resolution, Bradyrhizobium was the most dominant genera (Figure 2a). Compared to the control, AgNPs at higher concentrations increased the relative abundances of diazotrophic genera, including Bradyrhizobium, Anaeromyxobacteria and Geoalkalibacter. In contrast, AgNPs at 5 and 10 mg kg−1 decreased the relative abundance of Azospirillum (Figure 2a). AgNPs significantly affected the soil diazotrophic community composition, as reflected by PCoA and NMDS analyses. The soil diazotrophic community compositions were clustered into different groups according to the concentrations of AgNPs (Figure 2b,c).
To distinguish the significant changes of diazotrophic taxa, the effects of AgNPs on the diazotrophs at the genus level were further evaluated according to the response ratio method. The 95%CI of soil diazotrophs under 1 mg kg−1 and 5 mg kg−1 of AgNPs ranged from −0.04 to 0.04 and −0.05 to 0.07 and overlapped zero, which indicates that AgNPs at 1 mg kg−1 and 5 mg kg−1 did not bring significant effects on the diazotrophic community composition (Figure S3). AgNPs at 10 mg kg−1 and 20 mg kg−1 showed significant effects as the 95%CI of soil diazotrophs did not overlap zero. The relative abundances of Rhizobium, Methylomonas, Methylocystis, Syntrophobacter, Pelobacter, Azospirillum and Skermanella were decreased by AgNPs. In contrast, the relative abundances of Bradyrhizobium, Anaeromyxobacter and Methylobacter were increased by AgNPs (Figure 3).

3.4. Co-Occurrence Network Patterns of Soil Diazotrophic Community

Two co-occurrence networks were structured from AgNP and non-AgNP additions (Figure 4) to assess the effects of AgNPs on interspecific interactions. All the networks had modular structures, as reflected by the modularity values (greater than 0.4) [24]. Random networks were constructed and statistics of network matrices among different treatments were tested (Tables S2 and S3).
It was found that most network matrices were significantly different between treatments. Compared to the control, more edges and positive correlations were found in the AgNP treatment. The average degree, centralization degree and geodesic efficiencies were greater under AgNPs. By contrast, the average clustering coefficient, negative correlation and transitivity were lower under AgNPs (Table 3).
Based on the significance of connections among diazotrophic nodes, four nodes (Geobacter, Azospirillum, Burkholderia, Bradyrhizobium) and three module hubs (Bradyrhizobium, Paenibacillus, Skermanella) were classified as module hubs under control and AgNP treatments, respectively. Two nodes (Paenibacillus, Cupriavidus) were connectors under the control. In contrast, no nodes were connectors under AgNPs (Figure 5).

3.5. Linkages of Soil Diazotrophic Community to Plant and Soil Properties

Soil N2-fixation activity and nifH gene abundance significantly correlated with the soil pH, DOC and soluble Ag contents (Table S4). Observed OTUs and ACE also significantly correlated with the soil pH and soluble Ag content. The Mantel test found that the community composition of diazotrophs was significantly related to the available Ag and pH (Table 4). Soil N2-fixation activity and nifH abundance are negatively related to available Ag (Figure S4). Soil AK, AP and TN showed minor influences on diazotrophic activity, abundance and diversity (Table S4).
Soil soluble Ag, pH and DOC showed different impacts on the relative abundance of diazotrophic genera. Soil-soluble was Ag negatively related to the relative abundances of Rhizobium and Methylocystis, and positively related to Dysgonomonas and Anaeromyxobacter. Soil pH positively correlated with Methylocystis and negatively related to Anaeromyxobacter. DOC also has a positive correlation with Anaeromyxobacter. Moreover, Methylocystis negatively correlated with EC. Plant shoot biomass had positive correlations with the relative abundances of Azospirillum and Rubrivivax. In contrast, Soil AK and root biomass showed minor influences on the relative abundances of diazotrophic taxa. The relative abundances of Methylomonas and Methylocystis significantly correlated with available Ag. The relative abundance of Anaeromyxobacter was positively related to the soil pH (Figure 6).

4. Discussion

Assessment of the N2-fixation activity, abundance and community structure of diazotrophs is important for the mechanism exploration of the responses of N cycling to environmental changes and the linkages of belowground processes with plant changes [25]. In this study, AgNPs adversely affected the soil N2-fixation activity and nifH gene abundance (Figure 1). This result confirms our first hypothesis and is consistent with the common knowledge that AgNPs could be toxic to microbes due to their excellent antimicrobial properties. The biotoxicity of AgNPs is usually associated with both the released Ag ions and the nanoparticles themselves [26]. Here, it is exemplified that both the soil N2-fixation activity and nifH gene abundance were negatively correlated with the soil-soluble Ag content (Figure S4). However, no significant changes in the diazotrophic diversity were observed under AgNPs (Table 1). Thus, it can be postulated that the N2-fixation activity might be more sensitive to AgNPs relative to the diazotrophic diversity in maize soils. Jiang et al. [27] also found that the N2-fixation activity rather than diazotrophic diversity was significantly affected by heavy metals. Furthermore, the reduction in N2-fixation activity and nifH gene abundance might be also due to the decreased soil carbon bioavailability. AgNPs were documented to inhibit plant growth [14] and reduce root activity and exudates [28]. In the present study, a significant reduction in plant biomass and DOC contents was observed under AgNPs (Table 1). As the primary fuel for soil microbes, changes in carbon resources might alter the growth and activity of microorganisms. Consistently, the decreases in N2 fixation activity and nifH gene abundance positively correlated with the decreased soil DOC content (Table S4). In a previous investigation, Liu et al. [29] observed that both the N2-fixation activity and nifH gene abundance positively correlated with soil carbon content. Moreover, the reduction in N2-fixation activity positively correlated with the decreased nifH gene abundance (Figure 1), implying that the adverse effects of AgNPs on the nifH gene abundance could be transferred to the N2-fixation process.
Meanwhile, the soil diazotrophic community composition was strongly shifted by AgNPs, with dose-dependent changing patterns among different AgNP concentrations (Figure 2). Such phenomena are consistent with the case of bacterial and fungal communities under AgNPs [4,5] and diazotrophs under copper oxide NPs and heavy metals (e.g., cadmium, lead) [11,27]. According to the species-sorting concept, microbial communities could adjust their composition to adapt to changed environmental conditions [30]. In this study, the relative abundance bacteria of genus Bradyrhizobium (alpha-Proteobacteria) increased under AgNPs (Figure 2). Bradyrhizobium was the dominant genera in the diazotrophic community across different AgNP treatments in maize soils (Figure 2). Diazotrophs of this genera have excellent environmental adaptability that can adapt well to disrupted environments [31]. Unlike the Bradyrhizobium, the relative abundance of genus Azospirillum (alpha-Proteobacteria) was less abundant under AgNPs. These contrasting results imply that the coping mechanism of Bradyrhizobium was not commonly shared across diazotrophic taxa within a class. Moreover, the relative abundances of Heliobacterium, Methylomonas, Methylocystis, Syntrophobacter, Pelobacter and Rhizobium decreased under AgNP treatments (Figure 2). This result indicates that these taxa might have poor tolerances to AgNPs or the associated changed soil microenvironments [32]. Correlation analyses found that the relative abundance of Methylocystis negatively correlated with the soil DTPA-Ag content (Figure 6) but positively correlated with soil pH. Methylocystis can more efficiently drive N cycling under lower pH conditions than other diazotrophs [33]. Thus, it seems that the selection effect of soil pH on Methylocystis was offset by the negative effects of Ag. Rhizobium can supply N with plants by colonizing their roots [34]. Their decreased abundances imply that the N cycle in maize soil could be impacted by AgNP exposure.
The plant–microbe interaction might provide another explanation for the variation of diazotrophic community composition under AgNPs. As N fixation is an energy-consuming activity, AgNPs may intensify carbon source competition among diazotrophs when plants reduce carbon allocation to belowground through inhibition of plant growth and root activity [14]. Competition among diazotrophic taxa may lead to a loss of microbial diversity and/or more abundant diazotrophic members [35]. Here, some symbiotic diazotrophs, such as Bradyrhizobium, Geobacter and Paenibacillus, were more abundant in AgNP treatments (Figure 5), which can be indicative that these symbiotic diazotrophs would play a more important role in fixing N under AgNPs. Studies have found that plants tend to increase their dependency on symbiotic microbes to uptake more nutrients according to their growth demands under adverse conditions. For example, Qiu et al. [36] found that plants could intuitively select some symbiotic fungi with high nutrient-absorbing properties to resist stresses. In the case of diazotrophs, Geobacter can adapt well to unfavorable conditions and play important roles in symbiotic N-fixation processes [37]. Geobacter could effectively reduce Fe (III) to Fe (II) in anoxic environments, which plays an important role in the metabolism of nitrogenase [38]. Thus, it is conceivable that the positive responses of these diazotrophic groups to AgNPs might also be a coping mechanism of maize under AgNP stress.
Microbial co-occurrence networks had higher positive interactions under AgNPs than those of the control, indicating increased cooperation among diazotrophic species. The Stress Gradient Hypothesis points out that interspecific interactions will change from negative to positive in ecological communities with increasing stresses [39]. The higher geodesic efficiencies and lower average harmonic geodesic distances in the co-occurrence network under AgNPs also confirmed increased species–species dependencies [40]. Moreover, the higher transitivity value of the ecological network under AgNPs (Table 3) indicates stronger couplings [41] and weaker stability of the co-occurrence network [42]. Furthermore, the losses of the key module and connecter nodes in the AgNP network supported this assumption, as lower values mean a network was changed from robustness to perturbations [43]. The module hubs shifted from Bradyrhizobium, Geobacter, Azospirillum and Burkholderia to Bradyrhizobium, Paenibacillus and Skermanella under AgNPs (Figure 5). This indicates that different diazotrophs were selected as network keystones to support the whole community under AgNP and non-AgNP conditions. Paenibacillus has a high nitrogenase activity and tolerance to extreme conditions [44]. It can alleviate the phytotoxicity of heavy metal stress by immobilizing heavy metals via secreted flocculant [45]. Thus, it is not surprising that Paenibacillus was recognized as one of the keystones in the diazotrophic co-occurrence network under AgNPs. This result was anticipated because some diazotrophs functioning in metal resistance were more abundant in polluted soils [46].

5. Conclusions

This study reveals that soil diazotrophs were sensitive to AgNP exposure. AgNPs decreased the soil diazotrophic activity and abundances, and caused significant variations in their community compositions. AgNPs destabilized the soil diazotrophic ecological networks by leading to weakened negative associations among species and promoting interspecific interaction dependency. These findings extend our understanding of the biological consequences of AgNPs in soil diazotrophs and provide a scientific basis for comprehensive evaluation of the impacts of nanoparticles on ecosystems. Future studies over longer timescales will be necessary to evaluate the linkages of soil microbial communities with crop growth in response to AgNPs and to other nanoparticles due to the increasing application of nanoproducts.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15071601/s1, Figure S1: Sequences and OTUs (a) and relative abundances of dominant phyla (b) of soil diazotrophic community obtained by nifH gene; Figure S2: Relative abundances of diazotrophic community at order level. Others: the proportion of taxa with low abundance (<0.1%); Figure S3: Significant changes in the diazotrophic genera under 1 mg kg−1 and 5 mg kg−1 of AgNPs relative to control based on the response ratio method; Figure S4: Linear correlations of soil N2-fixation activity (a) and nifH gene abundance (b) versus soil DTPA-Ag content; Table S1: Sequences and OTU numbers of soil diazotrophic community under different concentrations of AgNPs; Table S2: Topological properties of empirical molecular ecological networks (MENs) of additional microbial communities and their associated random MENs; Table S3: T-test showing the significance of differences of topological properties between different treatments; Table S4: Correlations between soil diazotrophic activity, abundances, diversity indices and plant and soil parameters.

Author Contributions

Conceptualization, J.C.; methodology, H.C. and S.L.; software and validation, C.F.; formal analysis and investigation, S.L.; resources, J.C.; data curation, S.L. and C.F.; writing—original draft preparation, H.C.; writing—review and editing, J.C.; visualization, C.F.; supervision, J.C.; project administration, H.C. and J.C.; funding acquisition, H.C. and J.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Fujian Province (2019J01282), National Natural Science Foundation of China (32171758, 32360303) and the Startup Foundation for Introducing Talent of Jiangxi Agricultural University (9232308147).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Soil N2-fixation activity (a) and the nifH gene abundance (b) under different concentrations of AgNPs. Letters above bars indicate significant different analysis among treatments (p < 0.05).
Figure 1. Soil N2-fixation activity (a) and the nifH gene abundance (b) under different concentrations of AgNPs. Letters above bars indicate significant different analysis among treatments (p < 0.05).
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Figure 2. Relative abundance of the diazotrophic genera (a), community composition of soil diazotrophs under different concentrations of AgNPs based on NMDS (b) and PCoA (c).
Figure 2. Relative abundance of the diazotrophic genera (a), community composition of soil diazotrophs under different concentrations of AgNPs based on NMDS (b) and PCoA (c).
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Figure 3. Significant changes in the diazotrophic genera under 10 mg kg−1 and 20 mg kg−1 of AgNPs relative to control based on the response ratio method.
Figure 3. Significant changes in the diazotrophic genera under 10 mg kg−1 and 20 mg kg−1 of AgNPs relative to control based on the response ratio method.
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Figure 4. Co-occurrence networks of the soil diazotrophic community under non-AgNPs (a) and AgNPs (b). Red and blue lines represent positive and negative correlations between nodes, respectively.
Figure 4. Co-occurrence networks of the soil diazotrophic community under non-AgNPs (a) and AgNPs (b). Red and blue lines represent positive and negative correlations between nodes, respectively.
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Figure 5. Zi-Pi plots depict the topological roles of diazotrophic OTUs under non-AgNPs (a) and AgNPs (b).
Figure 5. Zi-Pi plots depict the topological roles of diazotrophic OTUs under non-AgNPs (a) and AgNPs (b).
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Figure 6. Correlations between the relative abundances of diazotrophic genera and soil properties and plant biomass. Ag: DTPA-Ag; Shoot: shoot biomass; root: root biomass; Brad: Bradyhizobium; Sker: Skermanella; Azos: Azospirillum; Sino: Sinorhizobium; Anae: Anaeromyxobacter; Geoa: Geoakalibacter; Syn: Syntrophobacter; Agro: Agrobacterium; Pseu: Pseudomonas; Paen: Paenibacillus; Geo: Geobacter; Rub: Rubrivivax; Dech: Dechloromonas; Heli: Helibacterium; Pelo: Pelobacter; Dys: Dysgonomonas; Tre: Treponema; Rhi: Rhizobium; Pal: Paludibacter; Mba: Methylobacter; Mmo: Methylomonas; Mcy: Methylocystis; Mce: Methylocella; *, p < 0.05; **, p < 0.01.
Figure 6. Correlations between the relative abundances of diazotrophic genera and soil properties and plant biomass. Ag: DTPA-Ag; Shoot: shoot biomass; root: root biomass; Brad: Bradyhizobium; Sker: Skermanella; Azos: Azospirillum; Sino: Sinorhizobium; Anae: Anaeromyxobacter; Geoa: Geoakalibacter; Syn: Syntrophobacter; Agro: Agrobacterium; Pseu: Pseudomonas; Paen: Paenibacillus; Geo: Geobacter; Rub: Rubrivivax; Dech: Dechloromonas; Heli: Helibacterium; Pelo: Pelobacter; Dys: Dysgonomonas; Tre: Treponema; Rhi: Rhizobium; Pal: Paludibacter; Mba: Methylobacter; Mmo: Methylomonas; Mcy: Methylocystis; Mce: Methylocella; *, p < 0.05; **, p < 0.01.
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Table 1. Soil properties in different concentrations of AgNP treatments.
Table 1. Soil properties in different concentrations of AgNP treatments.
Treatments0 mg kg−11 mg kg−15 mg kg−110 mg kg−120 mg kg−1
Soil pH8.49 ± 0.03 a8.49 ± 0.02 a8.47 ± 0.02 ab8.46 ± 0.03 ab8.41 ± 0.02 b
Soil EC165.7 ± 1.62 b181.7 ± 6.62 a195.0 ± 15.4 a180.2 ± 11.7 a183.8 ± 23.5 a
Soil TN1.11 ± 0.02 a1.09 ± 0.02 a1.09 ± 0.03 a1.05 ± 0.08 a1.07 ± 0.04 a
Soil AP10.3 ± 0.64 a10.1 ± 0.05 a9.92 ± 0.70 a9.70 ± 0.08 a9.47 ± 0.37 b
Soil AK246.8 ± 10.3 a209.7 ± 11.7 a216.1 ± 7.46 a230.4 ± 24.1 a222.8 ± 9.29 a
Soil DOC53.8 ± 2.20 a53.0 ± 1.15 a49.2 ± 3.77 ab50.4 ± 2.40 a42.7 ± 2.77 b
Soil DTPA-Ag0.11 ± 0.03 c0.14 ± 0.04 c0.89 ± 0.06 c2.54 ± 0.16 b5.67 ± 0.99 a
Lowercase letters in the same rows indicate significant different analysis among treatments at p < 0.05.
Table 2. Soil diazotrophic community diversity indices under different concentration of AgNPs.
Table 2. Soil diazotrophic community diversity indices under different concentration of AgNPs.
Treatments0 mg kg−11 mg kg−15 mg kg−110 mg kg−120 mg kg−1
Chao1705.2 ± 26.2 a727.1 ± 52.7 a735.7 ± 62.0 a712.0 ± 40.7 a767.2 ± 21.2 a
Observed OTUs594 ± 4.36 a603.7 ± 45.5 a593 ± 5.19 a587.3 ± 15.9 a634 ± 24.4 a
ACE695.0 ± 9.41 a720.4 ± 60.3 a702.7 ± 23.9 a704.6 ± 28.6 a755.9 ± 31.9 a
Shannon 3.52 ± 0.04 a3.40 ± 0.11 a3.46 ± 0.12 a3.53 ± 0.06 a3.54 ± 0.02 a
Simpson0.065 ± 0.03 a0.062 ± 0.003 a0.065 ± 0.001 a0.072 ± 0.011 a0.068 ± 0.009 a
Good’s coverage0.998 ± 0.0001 a0.998 ± 0.0001 a0.998 ± 0.0001 a0.999 ± 0.0002 a0.998 ± 0.0001 a
Same letters in the same rows indicate no significant changes among treatments at p < 0.05.
Table 3. Topological metrics of co-occurrence networks under AgNPs.
Table 3. Topological metrics of co-occurrence networks under AgNPs.
Network MetricsControlAgNPs
Number of nodes476469
Number of edges879936
Negative 563543
Positive316393
R square of power-law0.8280.818
Average degree (avgK)3.6933.991
Average clustering coefficient (avgCC)0.1870.139
Average path distance (GD)8.9397.751
Geodesic efficiency (E)0.1450.163
Transitivity (Trans)0.2150.155
Harmonic geodesic distance (HD)6.8866.121
Maximal degree1316
Centralization of degree (CD)0.0200.026
Maximal betweenness12,207.3420,779.31
Centralization of betweenness (CB)0.0990.181
Maximal stress centrality (CS)13.465.277
Maximal eigenvector centrality 0.3140.265
Centralization of eigenvector centrality (CE)0.3050.255
Density (D)0.0080.009
Connectedness (Con)0.5950.654
Efficiency0.990.99
Table 4. Relationships of soil diazotrophic community compositions with soil and plant properties as revealed by Mantel test.
Table 4. Relationships of soil diazotrophic community compositions with soil and plant properties as revealed by Mantel test.
Matrixrp
Shoot biomass−0.070.669
Root biomass−0.090.682
Soil pH−0.6220.051
Soil AK−0.0390.625
Soil AP0.2170.110
Soil TN−0.1350.787
Soil DTPA-Ag−0.6540.025
Soil EC−0.0720.675
Soil DOC0.0070.443
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Chen, H.; Li, S.; Fan, C.; Cao, J. Silver Nanoparticles Alter the Diazotrophic Community Structure and Co-Occurrence Patterns in Maize Rhizosphere. Agronomy 2025, 15, 1601. https://doi.org/10.3390/agronomy15071601

AMA Style

Chen H, Li S, Fan C, Cao J. Silver Nanoparticles Alter the Diazotrophic Community Structure and Co-Occurrence Patterns in Maize Rhizosphere. Agronomy. 2025; 15(7):1601. https://doi.org/10.3390/agronomy15071601

Chicago/Turabian Style

Chen, Hui, Siyao Li, Chengheng Fan, and Jiling Cao. 2025. "Silver Nanoparticles Alter the Diazotrophic Community Structure and Co-Occurrence Patterns in Maize Rhizosphere" Agronomy 15, no. 7: 1601. https://doi.org/10.3390/agronomy15071601

APA Style

Chen, H., Li, S., Fan, C., & Cao, J. (2025). Silver Nanoparticles Alter the Diazotrophic Community Structure and Co-Occurrence Patterns in Maize Rhizosphere. Agronomy, 15(7), 1601. https://doi.org/10.3390/agronomy15071601

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