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Article

Fertilization Strategies Regulate Soil Viral Diversity and Functional Potentials in Nutrient Cycling

by
Jian Xiao
1,2,
Chuan Liu
1,
Rui Wei
1,
Zhilai Chi
3,4,
Peng Zhang
5 and
Zhen Yu
1,2,*
1
Jiangsu Provincial University Key Laboratory of Agricultural and Ecological Meteorology, Key Laboratory of Ecosystem Carbon Source and Sink-China Meteorological Administration (ECSS-CMA), School of Ecology and Applied Meteorology, Nanjing University of Information Science & Technology, Nanjing 210044, China
2
State Key Laboratory of Climate System Prediction and Risk Management, Nanjing University of Information Science and Technology, Nanjing 210044, China
3
Fujian Provincial Key Laboratory of Ecological Impacts and Treatment Technologies for Emerging Contaminants, Key Laboratory of Ecological Environment and Information Atlas (Putian University) Fujian Provincial University, College of Environmental and Biological Engineering, Putian University, Putian 351100, China
4
Institute of Soil and Fertilizer & Resources and Environment, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, China
5
Liaoning Huikang Testing and Evaluating Technology Co., Ltd., Shenyang 110179, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(10), 2425; https://doi.org/10.3390/agronomy15102425
Submission received: 25 September 2025 / Revised: 10 October 2025 / Accepted: 17 October 2025 / Published: 20 October 2025
(This article belongs to the Section Soil and Plant Nutrition)
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Abstract

Soil viruses are increasingly recognized as key regulators of microbial communities and biogeochemical cycles, yet their responses to long-term fertilization strategies remain poorly characterized. We conducted a four-year pot experiment in subtropical China to evaluate how chemical fertilizer (CF), biochar (BC), and organic fertilizer (OF) application influenced soil viromes compared with an unfertilized control (CK) treatment. Metagenomic analyses recovered 1581 viral contigs with distinct community structures across treatments. Lytic viruses dominated overall, with higher proportions in BC and OF treatments, positively correlated with soil fertility indicators. Diversity indices indicated that BC and OF treatments significantly enhanced viral richness and evenness relative to CK and CF treatments, reflecting broader microbial host niches. Virus–host link predictions revealed expanded networks under BC and OF treatments, particularly with Pseudomonadota, Cyanobacteriota, and Acidobacteriota, suggesting amendment-specific viral regulation. Functional annotation showed that OF and BC application enriched viral KEGG categories related to metabolism, transport, and signal transduction. Moreover, BC and OF application promoted nitrate reduction, nitrogen fixation, and phosphorus mobilization. Together, these findings highlight organic amendments as critical drivers of soil viral diversity and functional potential, linking viromes dynamics to sustainable nutrient cycling in agroecosystems.

1. Introduction

Soil fertility management is a cornerstone of sustainable agriculture, as nutrient availability and soil health directly determine crop productivity and ecosystem resilience [1,2,3]. In intensive agroecosystems, fertilization strategies are particularly critical, with chemical fertilizers widely applied to sustain yields, while organic amendments such as biochar and manure are increasingly promoted for their capacity to improve soil quality, nutrient retention, and carbon sequestration [4,5,6,7]. Long-term input of chemical or organic fertilizers alters soil physicochemical properties, microbial communities, and nutrient turnover processes, ultimately influencing both agroecosystem productivity and environmental outcomes [8,9,10]. However, most research to date has focused primarily on soil chemistry and microbial dynamics, with comparatively little attention given to the role of viruses in agricultural soils under different soil fertility management [11,12].
Soil viromes are the most abundant biological entities on Earth, with estimates exceeding 1031 particles globally [13,14]. Although once regarded as passive pathogens, they are now recognized as key ecological regulators that shape microbial community structure, mediate horizontal gene transfer, and drive biogeochemical cycling [15,16,17]. Viruses influence microbial hosts through two major lifestyles: the lytic cycle, in which viruses replicate and lyse their hosts, and the lysogenic cycle, in which viral genomes integrate into host chromosomes and persist until induction [18,19,20]. These contrasting strategies have profound implications for soil processes. Lytic infections accelerate microbial turnover, releasing cellular necromass and organic matter that contribute to nutrient fluxes, whereas lysogeny promotes viral persistence and gene exchange, potentially equipping hosts with novel metabolic capabilities [21,22]. Shifts in viral lifestyle balance are closely tied to soil environmental conditions and host availability, yet remain poorly understood in managed agricultural soils.
In recent years, metagenomic analyses have revealed that viruses carry auxiliary metabolic genes (AMGs) that extend host functional capacity, especially in nutrient-limited environments [23,24]. In aquatic systems, viral AMGs have been shown to enhance photosynthesis, carbon fixation, and nitrogen transformations, thereby directly modulating ecosystem-level biogeochemical cycles [25]. Emerging evidence from soils indicates similar roles, with viral AMGs involved in organic matter degradation, nitrogen assimilation, and phosphorus acquisition [26,27,28]. For example, viral genes associated with phosphorus metabolism, such as phosphatases and transporters, have been identified in paddy soils [16,29], while nitrogen-related AMGs, including nitrate reductases and nitrogen fixation genes, have been reported in manure-amended fields [30]. Despite these advances, the ecological importance of soil viral functions in regulating nutrient cycling and sustaining agricultural productivity remains poorly understood. Soil viruses play crucial roles in the turnover of nitrogen and phosphorus, which are key determinants of soil fertility [16,26]. However, few studies have examined how long-term fertilization influences viral auxiliary metabolic genes and host–virus ecological networks. With the advent of high-throughput sequencing and the development of dedicated viral bioinformatics pipelines, it has become feasible to recover large numbers of viral contigs from complex soil metagenomes, to classify their taxonomy, to predict their functional repertoires, and to infer host associations with increasing precision [31,32]. Such approaches provide unprecedented opportunities to reveal the hidden roles of viruses in agroecosystems and their contributions to soil sustainability.
In this context, the present study provides one of the first metagenomic analyses linking fertilization regimes with viral diversity, lifestyle, host interactions, and nutrient cycling-related AMGs in a controlled, long-term pot experiment. The experiment included four treatments: unfertilized control, chemical fertilizer, biochar amendment, and organic fertilizer application. Specifically, we aimed to (i) characterize viral community diversity, taxonomic composition, and lifestyle strategies under different fertilization regimes; (ii) identify potential virus–host interactions and assess how organic versus inorganic amendments restructure viral ecological networks; (iii) evaluate the functional potential of soil viromes, with particular focusing on auxiliary metabolic functions related to nitrogen and phosphorus cycling. We hypothesize that organic amendments, including biochar and organic fertilizer, enhance soil viral diversity, expand host–virus associations, and enrich nutrient cycling–related AMGs compared with chemical fertilization, thereby promoting a more functionally resilient soil ecosystem. By linking viral dynamics with soil physicochemical properties and host ecology, our work provides new perspectives into the roles of viruses in agroecosystem nutrient cycling and highlights the ecological significance of fertilization strategies in shaping the soil virosphere.

2. Materials and Methods

2.1. Site Description

A long-term pot experiment was conducted in Nanjing, Jiangsu Province, China (32.04° N, 118.84° E) for four consecutive years. The region is under a northern subtropical humid climate characterized by a mean annual temperature of 15.4 °C, annual precipitation of 1106.5 mm, and approximately 117 rainy days per year.

2.2. Experiment Design

Each pot was filled with 10 kg of dry soils to a depth of 20 cm, and four fertilization treatments were established: (i) CK, unfertilized control; (ii) CF, chemical fertilizer (0.58 g N, 0.32 g P2O5, and 0.28 g K2O per pot); (iii) BC, chemical fertilizer inputs as in CF plus 100 g biochar produced from straw at 500–600 °C (particle size 0.002–2.0 mm, pH = 7.5); and (iv) OF, 50 g organic fertilizer derived from a composted mixture of sheep, cow, and pig manure. Nitrogen (urea) was applied both as basal and top-dressing fertilizer, while phosphorus, potassium, biochar, and organic fertilizer were supplied only as basal inputs. Each treatment included ten replicate pots to ensure experimental stability and reproducibility. Among them, three biological replicates were randomly selected for the determination of soil physicochemical properties and metagenomic sequencing to obtain representative viral community profiles while maintaining adequate sequencing depth per sample.

2.3. Soil Sampling

Soil samples were collected after the rice harvest in October 2024 to capture the cumulative effects of consecutive fertilization regimes. From each selected pot, four soil cores (0–20 cm depth) were taken at equidistant positions near the midpoints between the plant base and pot wall, and then thoroughly mixed to form one composite sample per pot. One portion was air-dried for the determination of physicochemical properties, while another portion was stored at −80 °C until molecular analyses.

2.4. Soil Physicochemical Properties Measurement

Soil pH was measured in a 1:2.5 (w/v) soil-to-water suspension using a calibrated pH meter. Soil organic matter (OM) and total nitrogen (TN) were quantified with an elemental analyzer (Vario EL III, Elementar, Hanau, Germany) after finely grinding the air-dried samples. Ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) were extracted with 2 M KCl (soil/solution = 1:5, w/v) and determined colorimetrically using a continuous flow analyzer (AA3, Seal Analytical, Hamburg, Germany). Total phosphorus (TP) was measured following H2SO4-HClO4 digestion, and available phosphorus (AP) was determined using the Olsen method with 0.5 M NaHCO3 extraction, followed by molybdenum–antimony colorimetric determination at 880 nm [33]. All measurements were performed in triplicate to ensure analytical accuracy, and results were expressed on an oven-dry soil weight basis.

2.5. DNA Extraction and Metagenomic Sequencing

Total microbial DNA was extracted from each soil sample using the PowerSoil® DNA Isolation Kit (QIAGEN, Germany) following the manufacturer’s protocol. DNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Library preparation was performed using the VAHTS Universal PLUS DNA Library Kit (Vazyme, China) with high-quality DNA following standard protocols. The prepared libraries were sequenced on the Illumina NovaSeq™ X Plus platform to generate 150 bp paired-end reads. Each sample was sequenced to a depth of 10 Gb per paired-end, yielding a total dataset of 240 Gb across all samples. The raw reads obtained in this study were submitted to the National Center for Biotechnology Information (NCBI) under the accession number PRJNA1338821. Raw sequencing reads were processed using Fastp (v0.20.1) for quality control [34]. This included adapter trimming, removal of low-quality bases, and filtering out reads containing ambiguous bases (N bases) to ensure high-quality data for downstream analysis.

2.6. Viromes Assembly and Functional Annotation

High-quality sequencing reads from all 12 samples were co-assembled using MEGAHIT (v1.2.9) with a min k-mer = 35, a max k-mer of 141, and a default k-step size to optimize assembly performance [35]. Viromes analysis was conducted using the ViWrap (v1.3.1) pipeline, a comprehensive framework integrating multiple tools for viral genome identification, classification, and characterization [36]. Specifically, VirSorter2 (v2.2.3) and VIBRANT (v1.2.1) were employed for automated viral genome detection and classification [37,38]. Only contigs ≥ 10 kb in length were retained for further analysis to ensure high-confidence viral genome reconstruction. Genome quality and completeness were assessed using CheckV (v1.0.1) to exclude low-quality sequences and minimize host contamination. To define non-redundant viral operational taxonomic units (vOTUs), viral genomes were clustered using dRep (v3.4.0) at 95% average nucleotide identity (ANI), and putative taxonomic relationships were further resolved with vContact2 (v0.11.0) based on shared protein content.
Viral taxonomy was assigned using homology searches against the NCBI RefSeq viral protein database combined with hidden Markov model (HMM)-based marker detection in HMMER (v3.3.2), with additional cross-validation using DIAMOND (v2.0.15) similarity searches. Putative host associations were predicted with iPHoP (v1.3.3), which integrates CRISPR-spacer matching, nucleotide similarity, k-mer composition, and machine learning-based classifiers, and host taxa were assigned within the GTDB (v2.3.2) phylogenetic framework [39,40]. Open reading frames (ORFs) were predicted with Prodigal, and functional annotation was performed using eggNOG-mapper (v5.0) with KEGG orthology assignments. Putative nitrogen- and phosphorus-related AMGs were identified by DIAMOND BLASTp searches against NCycDB (for nitrogen cycling) [41] and PCyCDB (for phosphorus cycling) [42]. The abundance of key metabolic pathways was quantified based on the annotated AMGs.

2.7. Statistical Analysis

Unless otherwise specified, all statistical analyses and visualizations were performed in R language (v4.4.1). Statistical differences were assessed using one-way ANOVA with Tukey’s HSD test, and different lowercase letters denote significance at p < 0.05. Viral community similarity among treatments was evaluated based on Bray–Curtis dissimilarities, followed by principal coordinates analysis (PCoA) and analysis of similarity (ANOSIM) using the vegan package. Homogeneity of multivariate dispersion among treatments was further evaluated using the betadisper function, and a permutation test (9999 permutations) was performed to confirm that differences in community composition were not driven by unequal variances. Alpha-diversity indices (Simpson and Chao1) were also calculated in vegan. To examine the relationships between viral communities and soil physicochemical properties, Procrustes analysis was performed using OmicStudio tools [43], and Mantel tests were then conducted with the linkET package. To identify the major drivers of viromes diversity, random forest modeling was carried out with the rfPermute (v2.5.5) package, and the relative importance of soil physicochemical variables was assessed based on %IncMSE with 1000 permutations. Model performance was evaluated using out-of-bag (OOB) R2 and root mean squared error (RMSE). Partial dependence plots (PDPs) were generated with the pdp package to visualize the marginal effects of key predictors on viral diversity [44,45]. Spearman’s rank correlations between the proportion of lytic viruses and soil physicochemical variables were calculated using the built-in corr function in R. For host-level analyses, Sankey diagrams were created in OmicStudio to illustrate virus–host interaction patterns. Functional profiles, including KEGG orthology assignments and comparisons of nitrogen- and phosphorus-related metabolic pathways, were visualized using the pheatmap package in R (v4.4.1).

3. Results and Discussion

3.1. Fertilization Strategies Restructure Soil Viral Community Composition and Diversity

A total of 1581 viral contigs were recovered across all treatments, representing the soil viromes shaped under different long-term fertilization regimes. The sequencing read counts for each sample and the summary statistics of viral contig length and completeness are provided in Tables S1 and S2, respectively. Principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarities revealed clear and significant separation of viral communities among treatments (ANOSIM: R = 0.89, p < 0.001) (Figure 1A). The first two PCoA axes explained 35.53% and 31.03% of the total variance, respectively. The beta-dispersion test indicated no significant differences in within-group variance (permutation test, p = 0.07), confirming that the observed separation reflects genuine compositional changes rather than differences in dispersion. Fertilization strategies strongly influenced soil viral community composition, consistent with previous findings that fertilization reshapes viromes through alterations in microbial hosts and soil environmental conditions [17,28,46]. Taxonomic classification showed that the majority of viral contigs (~75%) could not be assigned beyond the family level, which underscored the vast unexplored viral diversity in agricultural soils. Among classified viruses, the dominant families included Mimiviridae, Casjensviridae, Autographiviridae, Kyanoviridae, and Peduoviridae (Figure 1B). Notably, the relative abundance of Mimiviridae was higher in CK (3.45%) but declined to <1% in CF, BC, and OF treatments. In contrast, Tectiviridae, Mesyanzhinovviridae, and Peduoviridae were enriched in CF, BC, or OF treatments compared with other treatments, respectively. These patterns indicated that different fertilization inputs created distinct ecological niches, selectively favoring specific viral lineages, which may reflect shifts in host populations or nutrient-driven changes in viral life strategies [47,48]. Viral diversity indices further confirmed treatment-specific effects. Simpson diversity was significantly higher in BC and OF treatments compared with CK or CF treatment, whereas no significant differences were observed between CK and CF treatments or between BC and OF treatments (Figure 1C). In terms of richness, Chao1 estimates revealed that OF treatment harbored the highest viral richness, followed by CF and BC treatments, while CK treatment exhibited the lowest (Figure 1D). The elevated diversity and richness under BC and OF treatments highlight the role of organic amendments in sustaining more complex and resilient soil viromes. Biochar likely enhances the diversity by improving soil structure, buffering pH, and creating more heterogeneous microhabitats that sustain balanced host populations, while organic fertilizer provides abundant and diverse organic substrates that stimulate the microbial proliferation and expand the range of viral hosts [49,50].
Viruses in soils generally adopt either a lytic strategy, where they replicate and lyse host cells, or a lysogenic strategy, where they integrate into host genomes and persist until induction [14,18,28]. Across all treatments, lytic viruses predominated, accounting for 70.37–83.09% of the total viral community (Figure 2A). The proportion of lytic viruses was highest under biochar amendment (83.09%), followed by organic fertilizer (77.10%), while the lowest proportions occurred in CK and CF treatments (Figure 2B). This pattern suggests that organic inputs favor more active viral replication, whereas nutrient limitations under CK treatment and imbalanced inputs under CF treatment shift viruses toward lysogeny as a survival strategy [51]. Biochar likely promotes lytic activity by improving soil aeration, porosity, and nutrient buffering, which enhances microbial growth and host density [4]. Organic fertilizer similarly provides diverse carbon substrates and nutrients that sustain active microbial populations, thereby supporting viral propagation [5,6]. In contrast, unfertilized soils (CK) and chemical-only fertilization (CF) may restrict microbial productivity and induce conditions that favor viral dormancy.
Correlation analyses further supported these trends (Figure 2C). The proportion of lytic viruses showed significant positive associations with pH, OM, TN, TP, and AP (p < 0.05, r > 0.4), indicating that favorable soil fertility conditions promote lytic activity. Elevated organic matter supplies energy-rich substrates for microbial hosts, while higher N and P availability enhance microbial metabolism, creating opportunities for active viral infection cycles [16,51,52]. Soil pH, as a master regulator of microbial diversity and enzyme activity, may also indirectly influence viral strategies by shaping host communities [53]. These findings align with recent viromes studies in paddy soils, where long-term manure application increased the lytic-to-lysogenic ratio and enriched viral auxiliary metabolic genes [16], and contrast with poor environments where temperate (lysogenic) viruses dominate due to host scarcity and environmental stress [54,55]. From an ecological perspective, a shift toward lytic dominance under organic amendments has several implications [48]. It enhances microbial turnover, releasing necromass and dissolved organic matter that can contribute to soil organic carbon stabilization [19,32]. Viral lysis converts living microbial biomass into a pool of necromass rich in microbial cell wall fragments and extracellular polymers that are chemically recalcitrant and readily associate with mineral surfaces. This process, often referred to as the “viral shunt,” redirects a portion of microbial carbon from respiration to stable soil organic matter pools [56]. The released necromass can also fuel secondary microbial production, sustaining a dynamic cycle of carbon mineralization and stabilization. In this way, enhanced lytic activity may simultaneously accelerate short-term carbon turnover and promote long-term sequestration through the formation of mineral-associated organic carbon. In addition, lytic activity facilitates horizontal gene transfer, potentially accelerating the spread of functional traits, including nutrient-cycling genes and antibiotic resistance elements [16,57,58]. Furthermore, lytic viruses carrying nutrient-related AMGs can directly modulate nitrogen and phosphorus cycling, thereby linking viral life strategies to soil biogeochemical processes [16,28]. By contrast, lysogeny may dominate in nutrient-limited or stressed soils, enabling viral persistence but reducing immediate impacts on nutrient turnover [24].

3.2. Soil Physicochemical Factors Shape Viral Community Assembly and Diversity Patterns

To evaluate the relationships between viral community structure and soil properties, procrustes analysis was initially performed between the viral community matrix and the environmental factor matrix. The analysis showed a significant concordance (M2 = 0.175, p < 0.001) (Figure 3A), indicating that soil physicochemical factors strongly constrained viral community assembly. Similarly, Mantel tests further confirmed that viral richness (Chao1) was significantly correlated with multiple soil parameters, including OM, TN, NH4+-N, NO3−-N, TP, and AP, whereas viral diversity (Simpson) was less responsive, showing significant correlations only with OM and AP (Figure 3B). These findings suggest that soil fertility and nutrient pools exert greater influence on viral richness than on diversity, consistent with the notion that nutrient enrichment primarily expands the number of potential viral hosts and lineages [14,59].
To further disentangle the relative importance of individual factors, a random forest model was constructed using Chao1 and Simpson indices as response variables. For viral richness (Chao1), TN emerged as the most important predictor, followed by NO3−-N, TP, AP, and OM, whereas NH4+-N and pH exerted comparatively weaker effects (Figure 4A). Partial dependence plots (PDPs) were then constructed to visualize the relationships between soil factors and viral richness (Figure 4A). Specifically, an S-shaped response was observed between TN and viral richness. Chao1 index increased rapidly under low TN conditions, while plateaued once TN exceeded ~0.35%. A similar threshold effect was detected for TP, where viral richness increased with TP up to ~600 mg kg−1, beyond which further enrichment produced little additional effect. These saturation patterns indicate that viral richness is strongly limited by N and P pools in low-fertility soils, but becomes constrained by other factors once nutrient thresholds are surpassed. This aligns with previous work showing that nitrogen and phosphorus are primary drivers of viral proliferation, yet their effects are subject to diminishing returns at higher concentrations [16,60]. Importantly, our long-term treatments revealed that biochar and organic fertilizer substantially elevated TN, TP, and OM relative to CK and CF treatments, which likely explains why viral richness was consistently higher in BC and OF treatments. Biochar improves nutrient retention and soil buffering, thereby sustaining microbial hosts, while organic fertilizer supplies diverse and labile substrates that expand host niches [4,5,6]. Both inputs therefore push soils past critical nutrient thresholds, favoring expansion of viral lineages compared with chemical-only fertilization. For OM, NH4+-N, NO3−-N, and AP, the PDPs displayed the right-hand side of a U-shaped curve. At low nutrient levels, changes had only marginal effects on richness, but beyond certain thresholds, richness increased sharply. These nonlinear relationships suggest that viral richness is constrained under nutrient-poor conditions due to limited host activity, but once resource availability improves, microbial hosts proliferate, and viral communities expand rapidly. In particular, OM is a fundamental energy source that stimulates microbial biomass and activity, thereby sustaining viral diversification [14,61]; AP and NO3−-N provide readily utilizable nutrients that directly fuel microbial metabolism [62,63], while NH4+-N, though showing weaker effects in correlation analyses, contributes to host growth once available in sufficient concentrations.
For viral diversity (Simpson index), the random forest analysis ranked OM and AP as the strongest predictors, followed by TP and TN, while NO3−-N, NH4+-N, and pH exerted comparatively limited influence (Figure 4B). The prominent role of OM and AP reflects their direct contribution to host microbial productivity and resource heterogeneity: OM supplies carbon substrates that fuel microbial growth and broaden host niches, while AP represents the most bioavailable form of phosphorus, a critical element for nucleic acid synthesis and energy metabolism [61,64]. These two factors thus directly regulate host abundance and functional diversity, which in turn shape viral diversity. Similar patterns have been observed in soil bacterial communities, where OM and available phosphorus consistently emerge as key drivers of diversity under long-term fertilization [19,65,66]. Viral studies in agricultural soils also emphasize phosphorus as a central determinant of viral communities, with phosphorus-related AMGs frequently detected in viromes from P-enriched soils [26,28]. Partial dependence plots revealed the diverse response of environmental factors to viral diversity. For OM, diversity followed an S-shaped trajectory-remaining stable under low levels, sharply increasing at mid–moderate concentrations, and stabilizing again at higher concentrations. By contrast, AP and TP, as well as most other factors, exhibited saturating curves or diminishing return patterns: diversity increased sharply under nutrient-poor conditions but plateaued once nutrient availability exceeded a critical level. This indicates that viral diversity is most sensitive to changes under low-resource conditions but becomes less responsive when resources are abundant. The fact that OM and AP are consistently enhanced under biochar and organic fertilizer regimes explains why diversity indices were significantly higher in BC and OF than in CK or CF treatment (Table 1), underscoring the importance of organic amendments in promoting more balanced viral communities.
The distinction between richness and diversity is also notable. While viral richness (Chao1) responded strongly and linearly to multiple nutrients (especially TN, NO3−-N, and AP), viral diversity (Simpson) was mainly shaped by OM and AP and showed threshold-like responses. This suggests that richness is primarily controlled by the expansion of host niches with nutrient enrichment, whereas diversity is more sensitive to the balance among viral populations [67,68]. A likely explanation is that while more nutrients promote the emergence of additional viral taxa (higher richness), evenness stabilizes once dominant host populations become saturated, leading to limited further gains in diversity. Interestingly, these patterns parallel those observed in soil bacterial communities, where nutrient additions often increase species richness but have weaker or saturating effects on community evenness [69,70]. Similarly to bacteria, viruses may be constrained by strong host–virus specificity: once dominant host taxa flourish, their associated viruses can monopolize the community, preventing further increases in evenness despite additional nutrient inputs [48,58]. In this context, biochar and organic fertilizer appear to mitigate these constraints by enhancing resource heterogeneity and sustaining diverse microbial hosts, thereby promoting not only higher richness but also greater evenness compared with chemical fertilization alone [71,72]. This highlights the ecological significance of organic amendments in maintaining viral community complexity and stability in intensively managed farmland soils.

3.3. Organic Amendments Expand Virus–Host Networks and Reshape Functional Associations

To better understand viral ecological roles, we linked viral contigs to potential bacterial hosts and obtained 361 virus–host associations involving 256 viral contigs and 322 host contigs (Figure 5A). The associated viruses were primarily affiliated with Kyanoviridae, Mimiviridae, Mesyanzhinovviridae, and Peduoviridae, while their hosts were dominated by Pseudomonadota, followed by Cyanobacteriota, with Acidobacteriota and Chloroflexota also being common. The identified virus–host pairs exhibited varied infection efficiencies and relative abundances across different treatments, indicating that multiple viral lineages exploit different host taxa under distinct soil management regimes. Specifically, compared with CK treatment, CF treatment did not substantially increase virus–host connections, whereas organic amendments (BC and OF) significantly expanded viral–host associations compared with CK treatment (Figure 5B). Although CF treatment increased soil nutrient levels relative to CK treatment, it did not markedly restructure viral ecological networks, suggesting that the addition of inorganic nutrients alone is insufficient to promote extensive viral–host interactions. By contrast, the more diverse and heterogeneous resource inputs provided by biochar and organic fertilizer created favorable microhabitats that sustained richer microbial communities and broadened the range of potential viral associations. Notably, most of the additional links under BC and OF treatment were associated with Cyanobacteriota and Pseudomonadota (Figure 5C). Both phyla are functionally important in agricultural soils. Pseudomonadota include metabolically versatile bacteria central to nitrogen cycling processes, such as denitrification and nitrate reduction [73], while Cyanobacteriota contribute to carbon fixation and phosphorus turnover through photosynthetic activity and alkaline phosphatase production [74]. The enrichment of viral interactions with these phyla under BC and OF suggests that organic amendments stimulate the growth of functionally important microbial groups, thereby extending viral–host networks. Such dynamics are consistent with the “kill-the-winner” hypothesis, whereby the proliferation of fast-growing copiotrophic hosts (e.g., Pseudomonadota in BC-amended soils) triggers viral infections that prevent host overdominance, recycle host-derived organic matter, and enhance nutrient release [20,75,76]. In this way, viruses act not only as regulators of microbial community balance but also as mediators of nutrient turnover and agroecosystem stability under organic fertilization.
Interestingly, biochar favored more virus–Pseudomonadota associations, while organic fertilizer promoted stronger viral links with Cyanobacteriota. In addition, the number of viral connections with Acidobacteriota and Chloroflexota was also higher in OF treatment than in BC treatment. These differences reflect how the two organic amendments distinctly reshape soil physicochemical conditions and, consequently, microbial ecological niches. Biochar is well known to improve soil aeration, increase porosity, buffer pH, and enhance nitrate retention [4]. Such conditions preferentially support Pseudomonadota, a copiotrophic and metabolically versatile group that thrives in nutrient-enriched and oxygenated environments and plays key roles in nitrification, denitrification, and organic matter decomposition [50,77]. Viral infections targeting Pseudomonadota under BC treatment may therefore act as important regulators of nitrogen cycling, not only by controlling the abundance of fast-growing bacterial hosts but also by mediating the turnover of nitrogen-related AMGs. By contrast, organic fertilizer delivers a continuous influx of labile carbon and multi-nutrient inputs (N and P), creating a more heterogeneous and resource-rich environment that supports a wider array of microbial groups, including Cyanobacteriota, Acidobacteriota, and Chloroflexota. Cyanobacteriota contribute to carbon fixation and phosphorus mobilization, while Acidobacteriota, often considered oligotrophic taxa, exploit diverse organic substrates and help sustain microbial activity under fluctuating nutrient conditions [78,79]. Chloroflexota, in turn, are involved in organic matter decomposition and, in some cases, participate in anaerobic carbon cycling [80]. The greater number of virus–host connections with these groups under OF suggests that organic fertilizer not only enhances host abundance but also expands viral ecological networks across both copiotrophic and oligotrophic lineages. This expansion is likely due to the heterogeneous nutrient environment and diverse microhabitats created by manure-derived organic matter, which open ecological opportunities for a broader spectrum of viruses and their hosts [16]. These patterns demonstrate that organic amendments increase viral connectivity but also reprogram viral influence on soil biogeochemical processes. Biochar appears to channel viral regulation more narrowly toward nitrogen-associated bacterial hosts, leading to targeted impacts on nitrogen cycling, whereas organic fertilizer fosters broader virus–host associations across multiple phyla, thereby enhancing ecosystem multifunctionality by simultaneously influencing carbon, nitrogen, and phosphorus turnover. This amendment-specific restructuring of virus–host networks underscores the critical role of soil management practices in shaping not only microbial communities but also the ecological functions of their viral partners, ultimately affecting nutrient cycling and agroecosystem sustainability.

3.4. Long-Term Fertilization Alters Viral Functional Potential and Nutrient–Cycling Pathways

Viral functional annotation based on KEGG categories revealed that long-term fertilization markedly reshaped the metabolic potential of soil viromes (Figure 6A). Specifically, functions associated with replication and repair and nucleotide metabolism were substantially enriched in OF and BC, indicating more active viral replication under nutrient-rich conditions. This pattern suggests that organic amendments not only increase viral abundance but also accelerate viral turnover, consistent with the dominance of lytic viruses observed in these treatments. Similar enrichments have been reported in paddy soils, where manure inputs enhanced viral replication potential in parallel with stimulated host activity [16]. In addition, viral functions linked to carbohydrate metabolism, amino acid metabolism, and energy metabolism were strongly enhanced in OF and moderately enriched in BC. These pathways are central to host carbon and nitrogen metabolism, implying that viruses may provide auxiliary enzymes that accelerate organic matter decomposition and improve microbial nutrient acquisition [32]. The pronounced enrichment under OF treatment corresponds to elevated soil OM, TN, and TP, which create conditions favorable for microbial proliferation and, consequently, viral functional diversification. Moreover, categories related to membrane transport and signal transduction also increased in OF and BC treatment relative to CF and CK treatment. These functions suggest that viruses contribute to host adaptation by improving nutrient uptake and regulatory flexibility, thereby facilitating microbial responses to fluctuating nutrient conditions and promoting more stable community functioning [81,82]. Interestingly, lipid metabolism functions were more strongly represented in BC treatment than in OF treatment. This may be linked to biochar’s influence on soil structure and microhabitats, which could favor viral assembly and host membrane remodeling [83]. In contrast, OF-amended soils exhibited greater enrichment in xenobiotic biodegradation and metabolism, reflecting the diverse organic compounds introduced through manure [84]. This implies that viruses in OF-amended soils may extend host metabolic capacity to degrade and recycle complex organic matter, thereby strengthening the coupling between viral ecology and soil carbon turnover. In contrast, the narrower functional profile under CF treatment suggests that inorganic inputs alone are insufficient to drive broad viral functional diversification. The enrichment of xenobiotic degradation in OF-amended soils and lipid metabolism in BC-amended soils further highlights amendment-specific pathways through which viruses contribute to agroecosystem multifunctionality.
Previous studies have confirmed that soil viromes act as important drivers of geochemical cycling [16,26,29]. Therefore, we further examined viral functions associated with the N and P cycle. Specifically, denitrification-related functions were most abundant in CF-amended soils, followed by CK-amended soils, whereas BC- and OF-amended soils showed marked reductions (Figure 6B). This pattern indicates that inorganic fertilization strongly stimulated denitrification-associated viral functions, likely due to nitrate accumulation under long-term CF application [85]. In contrast, the reduced abundance of these functions in BC and OF treatment suggests that organic amendments may have redirected viral contributions toward alternative N-related pathways, thereby mitigating viral-driven denitrification processes and potential N losses. Assimilatory nitrate reduction and dissimilatory nitrate reduction were strongly enriched in BC-amended soils, with moderate signals in OF-amended soils but almost absent in CK- and CF-amended soils. These results indicate that biochar amendments created microhabitats and nutrient conditions favoring viruses linked to nitrate transformation, which can support microbial incorporation of nitrogen into biomass or facilitate anaerobic respiration. Viral AMGs associated with nitrate reduction may encode enzymes such as nitrate and nitrite reductases that supplement host respiratory chains, enhancing nitrate utilization efficiency and enabling microbes to sustain energy production under oxygen-limited conditions [16]. Such viral facilitation of nitrate reduction can accelerate nitrogen turnover by coupling host metabolism with viral replication, thereby intensifying local nitrogen cycling and microbial productivity. This enrichment aligns with the known ability of biochar to enhance nitrate retention and create microsites with variable oxygen status, thereby supporting more diverse nitrate-utilizing microbial hosts [86]. Nitrogen fixation functions also exhibited strong treatment-specific effects. Viral genes related to N fixation increased under all fertilization regimes compared with CK treatment, with the highest values observed in OF treatment, followed by CF and BC treatments. Functions associated with organic nitrogen degradation and synthesis were particularly abundant in CK-amended soils, but declined substantially in CF, BC, and OF-amended soils, respectively. This may reflect a reliance on recycling of organic N in nutrient-limited unfertilized soils, whereas fertilization reduced the relative importance of these pathways by providing more accessible inorganic N [87]. Nevertheless, both BC and OF treatments maintained moderate levels of viral organic N functions, suggesting a role for viruses in supporting microbial turnover of organic matter in amended soils.
Analysis of viral functional profiles also revealed distinct enrichment patterns in phosphorus-associated metabolic pathways under different fertilization regimes (Figure 6C). OF-amended soils exhibited the strongest signals across several pathways, including the pentose phosphate pathway, phosphonate and phosphinate metabolism, and the phosphotransferase system. These enrichments suggest that organic amendments expand viral contributions to phosphorus mobilization, enabling microbial hosts to access diverse P sources, including phosphonates and phosphorylated carbohydrates [28,88]. In addition, OF treatment maintained high levels of organic phosphoester hydrolysis, indicating enhanced viral roles in liberating phosphate from organic compounds. Viral AMGs involved in phosphonate metabolism likely encode C–P lyase complex subunits or related hydrolases that break the stable carbon–phosphorus bond, thereby releasing bioavailable phosphate [16,27]. This viral complementation allows microbial hosts to exploit otherwise recalcitrant organophosphorus compounds, enhancing P acquisition under nutrient-limited conditions. Through this process, viruses not only support host survival and replication but also accelerate phosphorus mineralization and recycling in soil ecosystems. BC treatment also showed the pronounced enrichment of viral functions related to phosphorus turnover, most notably oxidative phosphorylation and transporters. These results imply that biochar reshaped the soil viromes toward supporting microbial energy generation and nutrient acquisition, consistent with its known role in improving soil aeration, nutrient retention, and microbial activity [4,14]. Furthermore, BC treatment exhibited substantial enrichment of phosphonate metabolism, highlighting its potential to facilitate microbial utilization of otherwise recalcitrant P forms [89]. By contrast, CF showed narrower effects. Although some enrichment was observed in pyruvate metabolism and the pentose phosphate pathway, phosphorus-specific pathways such as phosphonate metabolism or phosphoester hydrolysis remained limited. This suggests that inorganic inputs provide soluble P directly, reducing selective pressure for viral contributions to alternative P cycling functions. These patterns demonstrate that organic inputs broaden viral functional repertoires linked to phosphorus cycling, promoting pathways for phosphorus mobilization, transformation, and transport, whereas chemical fertilization narrows functional contributions by relying primarily on direct inorganic P supply.

4. Conclusions

This study demonstrated that long-term fertilization strategies distinctly reprogrammed soil viromes in rice agroecosystems. Organic amendments, including biochar and organic fertilizer application, enhanced viral diversity, expanded virus–host networks, and enriched auxiliary metabolic functions supporting nitrogen retention and phosphorus mobilization. By contrast, chemical fertilization primarily stimulated viral denitrification functions, potentially exacerbating nitrogen loss. These contrasting patterns indicated that organic inputs fostered multifunctional viral roles in sustaining microbial productivity and nutrient turnover, while chemical inputs imposed narrower effects. Our results underscore that soil viromes are not passive bystanders but active participants in agroecosystem nutrient cycling. Incorporating organic amendments into fertilization regimes, therefore, holds potential to not only improve soil fertility but also to shape viral ecological functions that promote sustainability and resilience in agriculturally managed soils. In future research, field experiments across different soil types and climatic regions should be conducted to validate and extend these findings under real agricultural conditions. Such studies would help elucidate the generality of fertilization-induced viral responses and deepen our understanding of how soil viruses contribute to ecosystem nutrient cycling and agroecosystem sustainability at larger spatial scales.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15102425/s1, Table S1. Summary statistics of raw sequencing data. Table S2. General statistics of assembled viral genomes from soil viromes.

Author Contributions

Methodology, C.L. and R.W.; software, C.L., R.W. and P.Z.; validation, Z.C.; formal analysis, Z.C. and P.Z.; investigation, J.X., C.L., R.W. and Z.Y.; resources, Z.C.; data curation, J.X., C.L., R.W., P.Z. and Z.Y.; writing—original draft preparation, J.X. and Z.Y.; writing—review and editing, J.X. and Z.Y.; visualization, P.Z.; supervision, J.X. and Z.Y.; project administration, J.X. and Z.Y.; funding acquisition, J.X., Z.C. and Z.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Jiangxi Provincial Natural Science Foundation (20232BAB213083), the Modern Agricultural Research Collaborative Innovation Program of Jiangxi Province (JXSNKYJCRC202324), the Startup Fund for Advanced Talents of Putian University (2023124), the Natural Science Foundation of Fujian Province (2025J011035), the National Natural Science Foundation of China (42107027), and the Startup Foundation for Introducing Talent of NUIST (1133142401004).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

Author Peng Zhang was employed by the company Liaoning Huikang Testing and Evaluating Technology Co., Ltd. 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.

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Figure 1. Overview of soil viral communities under different long-term fertilization regimes. (A) Principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarities. (B) Taxonomic structure and composition of viral contigs at the family level. (C) Simpson diversity index of viral communities across treatments. (D) Chao1 richness estimates of viral communities across treatments. Statistical significance was determined by one-way ANOVA with Tukey’s HSD test; different lowercase letters indicate significant differences at p < 0.05. R2 denotes the proportion of variance explained by fertilization treatment in the one-way ANOVA.
Figure 1. Overview of soil viral communities under different long-term fertilization regimes. (A) Principal coordinates analysis (PCoA) based on Bray–Curtis dissimilarities. (B) Taxonomic structure and composition of viral contigs at the family level. (C) Simpson diversity index of viral communities across treatments. (D) Chao1 richness estimates of viral communities across treatments. Statistical significance was determined by one-way ANOVA with Tukey’s HSD test; different lowercase letters indicate significant differences at p < 0.05. R2 denotes the proportion of variance explained by fertilization treatment in the one-way ANOVA.
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Figure 2. Viral lifestyle and correlations with soil physicochemical properties. (A,B) Relative proportions of lytic and lysogenic viruses across fertilization treatments. (C) Spearman correlations between the proportion of lytic viruses and soil physicochemical parameters. Asterisks indicate significance levels (* p < 0.05; ** p < 0.01). Different lowercase letters indicate significant differences at p < 0.05.
Figure 2. Viral lifestyle and correlations with soil physicochemical properties. (A,B) Relative proportions of lytic and lysogenic viruses across fertilization treatments. (C) Spearman correlations between the proportion of lytic viruses and soil physicochemical parameters. Asterisks indicate significance levels (* p < 0.05; ** p < 0.01). Different lowercase letters indicate significant differences at p < 0.05.
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Figure 3. Relationships between viral communities and soil physicochemical properties. (A) Procrustes analysis showing concordance between viral community composition and soil physicochemical factors. (B) Mantel test results showing correlations between viral diversity indices and soil physicochemical parameters. Asterisks indicate significance levels (*** p < 0.001; ** p < 0.01; * p < 0.05).
Figure 3. Relationships between viral communities and soil physicochemical properties. (A) Procrustes analysis showing concordance between viral community composition and soil physicochemical factors. (B) Mantel test results showing correlations between viral diversity indices and soil physicochemical parameters. Asterisks indicate significance levels (*** p < 0.001; ** p < 0.01; * p < 0.05).
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Figure 4. Influence of environmental factors on viral diversity. (A,B) Contributions of individual environmental factors to viral diversity indices, followed by partial dependence plots illustrating the response of diversity to each factor. Blue lines show the modeled marginal effects of environmental variables, green lines depict smoothed trend curves, and red shading indicates the 95% confidence interval ranges. Model accuracy is expressed as out-of-bag (OOB) R2 and root mean squared error (RMSE).
Figure 4. Influence of environmental factors on viral diversity. (A,B) Contributions of individual environmental factors to viral diversity indices, followed by partial dependence plots illustrating the response of diversity to each factor. Blue lines show the modeled marginal effects of environmental variables, green lines depict smoothed trend curves, and red shading indicates the 95% confidence interval ranges. Model accuracy is expressed as out-of-bag (OOB) R2 and root mean squared error (RMSE).
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Figure 5. Predicted virus–host interactions under different soil management regimes. (A) Sankey diagram displaying the connections between viral families and their bacterial hosts at the phylum level. (B) Distribution of virus–host associations across treatments presented as a pie chart. (C) Counts of predicted host phyla. Color assignments for host groups are kept consistent with the taxonomy scheme illustrated in panel (A).
Figure 5. Predicted virus–host interactions under different soil management regimes. (A) Sankey diagram displaying the connections between viral families and their bacterial hosts at the phylum level. (B) Distribution of virus–host associations across treatments presented as a pie chart. (C) Counts of predicted host phyla. Color assignments for host groups are kept consistent with the taxonomy scheme illustrated in panel (A).
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Figure 6. Functional comparison of soil viral communities under different fertilization treatments. (A) Relative abundance of viral functions across KEGG level 2 categories. (B) Predicted viral auxiliary metabolic functions associated with nitrogen cycle processes. (C) Predicted viral auxiliary metabolic functions related to phosphorus cycle processes.
Figure 6. Functional comparison of soil viral communities under different fertilization treatments. (A) Relative abundance of viral functions across KEGG level 2 categories. (B) Predicted viral auxiliary metabolic functions associated with nitrogen cycle processes. (C) Predicted viral auxiliary metabolic functions related to phosphorus cycle processes.
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Table 1. Soil physicochemical properties under contrasting long-term fertilization treatments.
Table 1. Soil physicochemical properties under contrasting long-term fertilization treatments.
TreatmentpHOM (%)TN (%)NH4+-N
(mg/kg)		NO3−-N (mg/kg)TP
(mg/kg)		AP
(mg/kg)
CK6.34 ± 0.13 b2.50 ± 0.24 c0.19 ± 0.01 b17.32 ± 0.59 b0.96 ± 0.09 c401.63 ± 17.41 c26.80 ± 2.45 c
CF6.34 ± 0.18 b2.40 ± 0.04 c0.19 ± 0.02 b22.19 ± 1.75 a1.93 ± 0.11 b419.37 ± 28.89 c26.49 ± 1.03 c
BC7.16 ± 0.20 a3.88 ± 0.25 b0.23 ± 0.02 b17.34 ± 0.95 b1.22 ± 0.10 c516.37 ± 32.86 b42.57 ± 2.46 b
OF6.44 ± 0.18 b4.33 ± 0.33 a0.37 ± 0.03 a23.41 ± 1.16 a2.98 ± 0.11 a659.61 ± 53.40 a51.13 ± 2.39 a
Note: Values are presented as mean ± SD (Standard Deviation). Different lowercase letters indicate significant differences at p < 0.05 for each parameter.
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Xiao, J.; Liu, C.; Wei, R.; Chi, Z.; Zhang, P.; Yu, Z. Fertilization Strategies Regulate Soil Viral Diversity and Functional Potentials in Nutrient Cycling. Agronomy 2025, 15, 2425. https://doi.org/10.3390/agronomy15102425

AMA Style

Xiao J, Liu C, Wei R, Chi Z, Zhang P, Yu Z. Fertilization Strategies Regulate Soil Viral Diversity and Functional Potentials in Nutrient Cycling. Agronomy. 2025; 15(10):2425. https://doi.org/10.3390/agronomy15102425

Chicago/Turabian Style

Xiao, Jian, Chuan Liu, Rui Wei, Zhilai Chi, Peng Zhang, and Zhen Yu. 2025. "Fertilization Strategies Regulate Soil Viral Diversity and Functional Potentials in Nutrient Cycling" Agronomy 15, no. 10: 2425. https://doi.org/10.3390/agronomy15102425

APA Style

Xiao, J., Liu, C., Wei, R., Chi, Z., Zhang, P., & Yu, Z. (2025). Fertilization Strategies Regulate Soil Viral Diversity and Functional Potentials in Nutrient Cycling. Agronomy, 15(10), 2425. https://doi.org/10.3390/agronomy15102425

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