Limited resilience of the soil microbiome to mechanical compaction within four growing seasons of agricultural management

Modern agriculture relies on heavy machinery, frequently causing soil compaction, especially under wet conditions. Compaction alters soil structure (increasing bulk density, reducing macroporosity and pore connectivity) and thereby reduces water and gas transport, affecting chemical reactions and biological processes. While physical impacts are well documented, consequences for soil microbiomes in arable systems are less understood; prior work has largely focused on biomass and specific functions, often at coarse taxonomic resolution, and high-throughput sequencing studies have been mostly in forests. The aim was to assess the impact of a single soil compaction event on soil microbial diversity and its temporal evolution over four growing seasons under different agricultural management systems (permanent ley, no-till rotation, conventional till rotation). The authors hypothesized that compaction would promote anaerobically respiring prokaryotes and saprotrophic fungi and reduce aerobically respiring prokaryotes and plant-associated fungi; they expected substantial short-term impact with largely recovering microbial communities over four seasons, and faster recovery in crop rotations, particularly with tillage, compared to permanent ley.

The paper reviews known effects of compaction on soil physical properties (increased bulk density and mechanical resistance, reduced macroporosity and pore connectivity), leading to reduced water infiltration, drainage, aeration, and altered chemical reactions dependent on oxygen and water. Biological consequences include reduced root growth and nutrient access, lower microbial biomass, and shifts from aerobic to anaerobic processes (e.g., decreased nitrification/mineralization, increased methanogenesis/denitrification), influencing greenhouse gas fluxes. Prior microbiome studies used PLFA or t-RFLP with limited taxonomic resolution; recent sequencing-based studies in forests showed compaction-driven increases in anaerobes and saprotrophs and incomplete resilience. However, forest results may not translate directly to arable systems due to differing management, highlighting a gap addressed by this study.

Study site and design: The Soil Structure Observatory (SSO) at Agroscope, Zurich, Switzerland (47.4°N, 8.5°E; 444 m a.s.l.) was established in 2014 on a pseudogley with temporary waterlogging and slight block-wise differences in texture, organic C, pH, and water table. The site was sown with a grass–legume ley in spring 2013. In April 2014, a single compaction event was applied using a two-axle self-propelled vehicle (wheel load 8 Mg, 1050/50R32 tires, 300 kPa inflation). Treatments: (1) control (no compaction), (2) tracks (three vehicle passes yielding six 1 m-wide wheel tracks per plot), and (3) areal (compaction of entire plot). Post-compaction management systems: permanent ley (PL), crop rotation with no tillage (NT), and crop rotation with conventional tillage (CT; moldboard plow to 0.25 m, rotavator to ~0.06 m), established eight days after compaction. Each combination of 3 compaction treatments × 3 management systems was replicated in three blocks (strip-plot; 36 plots of 16 × 12 m; BS not reported here). Management: PL was cut 4–5 times per year with manual removal; no traffic/tillage. Crop rotations: triticale (2014), silage maize (2015), winter wheat (2016), winter rapeseed (2017); standard fertilization (GRUDAF) and integrated pest management; seeding via no-till drill in NT; limited routine traffic uniformly across treatments. Physical sampling and measurements: Soil cores (0–20 cm) collected fall 2013 (pre), spring 2014 (weeks after compaction), spring 2015, 2016, 2017. Three undisturbed 100 cm³ cores per block × compaction × management at each time. Bulk density quantified after drying at 105 °C. Air permeability measured at 2 hPa overpressure; gas diffusivity measured using O2 in steady-state one-chamber apparatus. Physical properties reported for 2014 (immediately after) and 2017 (year 3 after) at 300 hPa matric suction. Yield and biomass: PL biomass (dry matter) harvested within two 0.5 × 0.5 m frames per plot (2014, 2017). Crop yields measured per season (grain for triticale, wheat, rapeseed; dry above-ground biomass for maize). Expressed as percent relative to uncompacted CT reference. Microbial sampling and extraction: Pre-compaction sampling in fall 2013 and spring 2014 at evenly spaced locations (n=104 total). Post-compaction sampling in fall 2014, spring 2015, 2016, 2017. For each time point, bulk soil (0–20 cm) from four 2 cm-diameter cores pooled at three random positions per block × treatment × management (81 samples per time). DNA extracted from 0.5 g soil via bead-beating protocol (Bürgmann et al.), purified (NucleoSpin), quality-checked, quantified (PicoGreen), adjusted to 5 ng/µl with BSA and heat treatment. Amplicon sequencing: Prokaryotes: 16S rRNA gene V3–V4 (primers 341F/806R). Fungi: ITS2 (primers ITS3ngs/ITS4ngs). PCR in technical triplicates on 40 ng DNA, pooled; barcoded via Fluidigm Access Array; sequenced paired-end on Illumina MiSeq v3. Average reads: prokaryotes 19,020 ± 4,700; fungi 20,225 ± 6,005. ENA accession PRJEB43264. qPCR for functional genes: Tested inhibition with PGEM-T plasmid spike (SP6/T7 primers). Standard curves from pooled amplified targets (10⁻¹ to 10⁷ ng DNA/reaction). Targets: 16S rRNA gene (515F/806R), nirK (5F/890F), nirS (CD3aF/R3cd). Reaction conditions provided; efficiencies: 16S 97.4% (R²=0.999), nirK 99.5% (R²=0.998), nirS 94.8% (R²=0.995). Copy numbers computed using Avogadro’s number and estimated amplicon sizes. nirK and nirS normalized by 16S copies. Bioinformatics: Pipeline based on VSEARCH. Primer trimming with CUTADAPT (≤1 mismatch), PhiX filtering (Bowtie2). Paired-end merging and quality filtering (max expected error=1). Dereplication and ASV delineation via UNOISE (alpha=2, minsize=4), chimera removal via UCHIME2. Ribosomal signature validation with Metaxa2 (16S) and ITSx (ITS2); non-supported sequences removed. Read mapping to verified ASVs at ≥97% identity, maxhits=1. Taxonomy via SINTAX against SILVA v132 (16S) and UNITE v7.2 (ITS2), bootstrap cutoff 0.8; non-bacterial/archaeal/fungal and organelle ASVs removed. Statistics: R-based analyses (p<0.05). Yields: PL differences by Kruskal-Wallis; crop rotation yields by ANOVA with Tukey HSD. Physical properties: Kruskal-Wallis with Dunn’s post hoc. Diversity: sequencing depth assessed by rarefaction; alpha diversity (observed richness, Pielou’s evenness, Shannon) and beta diversity (Bray-Curtis) from ten iteratively subsampled, square-root transformed ASV tables. Effects of block, date, treatment, management on alpha and beta diversity via PERMANOVA (adonis, 999 perms) with pairwise tests (RVAideMemoire). Ordinations: PCoA and CAP (BiodiversityR) with reclassification success. Taxon-level responses to compaction over time via PERMANOVA with spatial partitioning; multiple testing controlled via q-value (<0.05); rare/infrequent ASVs (overall abundance <0.5% and/or <4 samples) removed. Taxonomic trees visualized in iTOL; ecological inferences supported by literature, FAPROTAX (prokaryotes) and FUNGuild (fungi).

• Soil physical properties: Compaction increased topsoil bulk density by ~10–15% and reduced air permeability by 60–94% and gas diffusion by 48–66% shortly after the event. After four growing seasons, PL and NT did not fully recover in bulk density and still tended to show lower gas transport; CT largely recovered after mechanical loosening in spring 2014.
• Yields: PL biomass was ~19% lower in 2014 and ~32% lower in 2017 in compacted plots vs control (not statistically significant due to block variability). In rotations under NT, triticale (2014) and maize (2015) yields were ~51–58% of uncompacted CT in tracks and ~8–35% in areal compaction. Under CT, yields were at least 79% (triticale, 2014) and 90% (maize, 2015) of the uncompacted CT, with marginal effects in 2016–2017. Abstract notes yield reductions up to -90% in first two seasons with recovery thereafter.
• Alpha diversity: Prokaryotic richness, evenness, and Shannon diversity showed no compaction or management effects; fungal richness increased under areal compaction, while NT had lower fungal alpha diversity than PL and CT. Temporal and spatial (block) differences were significant, with lower prokaryotic alpha diversity in 2014–2015 and lower values in block A.
• Beta diversity: Microbial community structure differed significantly by compaction treatment and management (PERMANOVA). Explained variance: compaction ~1% (prokaryotes) and 2% (fungi); management 2% (prokaryotes) and 7% (fungi). Strong spatial block effects (15–17%) and temporal effects (3–8%). CAP reclassification success rates indicated clear discrimination, strongest in PL (prokaryotes 92–97%, fungi 89–100%), intermediate in NT (prokaryotes 89–92%, fungi 94–97%), and lowest in CT (prokaryotes 72–86%, fungi 88–97%). No significant interaction with time indicated little resilience over four seasons.
• Compaction-sensitive taxa: ~24% of 3,871 prokaryotic ASVs and ~43% of 1,141 fungal ASVs responded to compaction (q<0.05), mostly management-specific. Anaerobic/oxygen-limited taxa increased (e.g., Geobacter, Desulfuromonas, Anaeromyxobacter, Anaerolinea, Longilinea, Dechlorosoma, Methanosarcina; fungi Mortierella, Mucor, Tetracladium, Preussia, Botryotrichum, Scutellinia, Thelebolus). Aerobic and plant-associated taxa decreased (e.g., Nitrospira, Mycobacterium, Demequina, Pseudomonas, Bacillus, Candidatus Nitrososphaera; fungi Glomus, Trichoderma, Aspergillus, Penicillium, Ustilago). No significant time-dependent recovery of responding genera.
• Functional genes: nirS and nirK (normalized to 16S) tended to increase under compaction in PL and NT in 2014 and decrease under CT, consistent with impaired aeration; differences were not statistically significant due to block variability.
• Overarching: Soil microbial community structure exhibited limited resilience compared to partial recovery of physical properties and yields; strong spatial heterogeneity influenced outcomes.

The study shows that a single mechanical compaction event can cause long-lasting changes in soil physical properties and microbial community structure in arable topsoil, with only partial recovery over four growing seasons. While crop yields largely recovered after the first two years (especially under CT), microbial beta diversity remained significantly shifted, indicating limited structural resilience of the microbiome. Findings support the hypothesis that reduced pore space and oxygen diffusion favor anaerobic prokaryotes and saprotrophic fungi, while aerobic nitrifiers and plant-associated microbes decline, potentially affecting nitrogen cycling (e.g., reduced nitrification) and plant–microbe interactions. Differences among management systems indicate that CT can mitigate topsoil physical impacts and attenuate microbiome shifts relative to PL and NT, though CT also showed distinct community structures. Spatial heterogeneity (block effects) and interannual variability were dominant drivers, underscoring the importance of initial edaphic conditions (texture, pH, organic C) and climate (e.g., drought legacy) in modulating compaction effects. The lack of observed microbiome resilience compared with partial physical and yield recovery suggests that microbial community legacies might persist beyond physical metrics, raising concerns for cumulative impacts in regularly trafficked fields.

A single compaction event produced persistent alterations in soil properties and microbiome composition across four growing seasons. Compaction consistently increased anaerobic and saprotrophic taxa and reduced aerobic and plant-associated microorganisms, aligning with oxygen limitation due to decreased pore connectivity. Microbial community structure showed limited resilience despite some recovery of soil gas transport properties and crop yields, especially under CT. These results highlight the risk of cumulative microbiome and functional impacts from repeated compaction in conventional operations. Future research should quantify cumulative and long-term legacy effects of repeated compaction on the microbiome and biogeochemical functions, evaluate links between microbial shifts and agroecosystem productivity and nutrient cycling, and develop management strategies (e.g., traffic control, optimized tillage, cover crops, biological remediation) that minimize compaction impacts while sustaining soil function.

• Strong spatial heterogeneity across replicated field blocks (pre-existing differences in texture, pH, organic C, water table) led to variable compaction intensity and limited statistical power for some responses (e.g., yields, gas transport, nir genes). - Microbial functional inference based on taxonomic identity and guild databases has constraints and may miss relevant traits. - Bare soil (BS) treatment was not reported here. - Gene abundance (nirS/nirK) trends were not statistically significant, possibly due to block variability. - Analyses focused on topsoil (0–20 cm); subsoil impacts and persistence may differ. - Four growing seasons may be insufficient to capture full long-term microbiome recovery trajectories or weather-driven variability.