Modern agricultural mechanization, while enhancing efficiency, often leads to soil compaction, particularly during operations under unfavorable soil conditions. The recovery of compacted soil can take decades without proper management, posing a significant threat to soil health and ecosystem functioning. Soil compaction negatively alters soil structure, increasing bulk density and mechanical resistance, while simultaneously reducing macroporosity and pore connectivity. This results in impaired water and gas transport, hindering water infiltration and drainage, and reducing soil aeration. These changes also influence soil chemical reactions dependent on oxygen and water availability. The topsoil is considered the most sensitive layer due to its high concentration of root biomass, microbial biomass, and diversity, and is the preferred habitat for many soil fauna. Compaction affects soil biology directly (e.g., increased resistance to bioturbation) and indirectly (e.g., altered oxygen and moisture levels). Increased mechanical resistance limits root elongation and rooting depth, reducing water and nutrient access and consequently impacting crop yield. Pore space limitation restricts microbial access and can reduce soil microbial biomass. Aerobic processes like nitrification and mineralization are negatively affected due to decreased macropores and oxygen diffusion, potentially favoring anaerobic microorganisms. Changes in microbial community composition and activity can alter carbon and nitrogen metabolism, reducing basal respiration and increasing methanogenesis and denitrification. While the impacts of compaction on physical soil properties are relatively well understood, knowledge about its consequences for the soil microbiome is limited, particularly in arable agricultural systems. High-throughput DNA sequencing offers a powerful tool to address this gap. This study aimed to assess the impact of soil compaction on soil microbial diversity and its temporal evolution under different agricultural management systems over the first four growing seasons after a single compaction event. This links knowledge of changes in soil physicochemical properties and crop yield to the ecosystem functions mediated by microorganisms in arable fields. The study hypothesized that compaction would alter microbial community structures, favoring anaerobically respiring prokaryotes and saprobic fungi while limiting aerobically respiring prokaryotes and plant-associated fungi. It also predicted a short-term impact with recovery over four growing seasons, with faster recovery in crop rotations with tillage operations compared to a permanent ley.

Existing literature highlights the detrimental effects of soil compaction on various aspects of soil health and agricultural productivity. Studies have documented the long-term persistence of compaction, with recovery times often spanning decades. The European Commission and FAO recognize soil compaction as a major threat to soil resources, emphasizing the need for assessment and appropriate regulations. Research has extensively shown the negative impacts of compaction on soil structure, leading to increased bulk density, reduced porosity, and impaired water and gas transport. The consequences for soil organisms are significant, impacting root growth, microbial biomass and activity, and the overall functioning of the soil ecosystem. While some studies have investigated the effects of compaction on microbial biomass and specific functional activities like greenhouse gas fluxes, detailed analyses of microbial diversity using high-throughput techniques have been limited, particularly in agricultural settings. Previous research in forest ecosystems has provided some insights, but these findings are not directly transferable to arable agricultural systems due to fundamental differences in management practices. This lack of data on microbial diversity in compacted arable soils makes this study crucial for understanding the long-term impacts of compaction on soil ecosystems.

This study utilized data from a long-term Soil Structure Observatory (SSO) established in 2014. The experimental design involved three compaction treatments: a control (no compaction), compaction in wheel tracks ('tracks'), and compaction of the entire plot area ('areal'). These were combined with four post-compaction agricultural management systems: permanent ley (PL), bare soil (BS), crop rotation under no tillage (NT), and crop rotation under conventional tillage (CT). Each combination was replicated three times in a strip-plot design across three blocks. Soil physical properties (bulk density, air permeability, gas diffusion) were measured before compaction and at various time points afterward. Crop yield (grain weight or above-ground biomass) was assessed annually. Microbial community structure and gene abundance were analyzed using high-throughput DNA sequencing targeting bacterial and archaeal 16S rRNA genes and fungal ITS2 regions. Samples were collected before and at several intervals after compaction. DNA extraction and purification were performed, followed by PCR amplification and sequencing on an Illumina MiSeq platform. Bioinformatic analysis using a custom pipeline involved primer trimming, quality filtering, merging, dereplication, ASV delineation, chimera removal, and taxonomic classification using SILVA and UNITE databases. Functional gene quantification (nirS, nirK, 16S rRNA) was done via qPCR. Statistical analyses in R included Kruskal-Wallis tests, ANOVA, PERMANOVA, and pairwise tests to assess differences in physical properties, crop yields, α-diversity (richness, evenness, Shannon diversity), β-diversity (Bray-Curtis dissimilarity), and taxonomic responses to compaction.

Soil compaction significantly increased bulk density (10-15%) and reduced air permeability (60-94%) and gas diffusion (48-66%) in PL and NT, with incomplete recovery within four seasons. CT mitigated compaction effects. Ley biomass was reduced in compacted plots, but arable crop yields largely recovered after the first two seasons. Microbial α-diversity (prokaryotes) showed no compaction effects; fungal richness increased under 'areal' compaction. β-diversity analysis revealed significant effects of agricultural management systems, with compaction explaining a smaller portion of the variance. Each compaction treatment and management system had distinct microbial communities. Compaction effects on microbial community structure depended on the management system, with the strongest differences observed in PL and less pronounced in CT. Spatial and temporal variability were major drivers of microbial community structure, with significant differences between blocks and years. Approximately 24% of prokaryotic and 43% of fungal ASVs responded significantly to compaction. Many sensitive ASVs were management system-specific. Anaerobic prokaryotes (e.g., *Geobacter*, *Anaeromyxobacter*, *Methanosarcina*) and saprotrophic fungi increased under compaction, while aerobic prokaryotes (e.g., *Nitrospira*, *Candidatus Nitrososphaera*) and plant-associated microorganisms decreased. qPCR analysis of functional genes (nirS, nirK) showed a tendency for increased abundance under compaction in PL and NT in 2014, but not in CT or in 2017. These changes, however, were not statistically significant due to high variability across blocks.

This study demonstrates that a single soil compaction event can have lasting effects on soil physical properties and the soil microbiome, even after four growing seasons of various management practices. The observed decrease in crop yield, particularly in the early years, can be attributed to the significant changes in soil physical properties, which limited root growth and nutrient uptake. The changes in microbial community composition, with the relative increase of anaerobic prokaryotes and saprotrophic fungi and a decrease in aerobic and plant-associated microorganisms, are consistent with previous research in forest ecosystems, supporting the idea that changes in soil aeration and plant health are major drivers of these shifts. The lack of full recovery of soil physical properties and microbial community structure highlights the long-term consequences of soil compaction. The high spatial variability underscores the influence of pre-existing conditions on the response to compaction. These results have significant implications for sustainable agricultural practices, emphasizing the need to manage soil compaction carefully to mitigate long-term negative impacts on soil health and productivity.

This study reveals the significant and persistent alteration of the soil microbial community structure by a single soil compaction event. The lack of resilience despite subsequent management interventions, and the consistent favoring of anaerobic and saprotrophic microorganisms are notable findings. The results emphasize the need for strategies to prevent soil compaction and potentially consider the long-term microbial consequences in future research on sustainable cropping systems. Further research should focus on the cumulative impacts of repeated compaction events, the functional implications of the observed shifts in microbial communities, and the effectiveness of different management practices in restoring soil health.

The high spatial variability observed across the three blocks, possibly due to pre-existing field heterogeneity, limited the statistical power of some analyses. The study focused on a single compaction event and four growing seasons, limiting the ability to fully characterize long-term dynamics. The conclusions regarding microbial lifestyles are based on taxonomic assignments and available literature, which might not capture the full complexity of microbial interactions. Further investigation into the functional roles of the identified taxa would strengthen the interpretation of the observed responses to compaction.