This research was conducted at the Conservation Agriculture Systems Innovation (CASI) facility in Five Points, California (36.34° N, 120.12° W). The site features Panoche clay loam soil (fine-loamy, mixed, superactive, thermic Typic Haplocambids) with a textural gradient from clay loam to sandy clay loam. The Mediterranean climate averages 178 mm annual precipitation and temperatures ranging from 7.8–23.9 °C. Complete site characterization is available in . The experiment employs a randomized complete block design with four treatments (combining standard or reduced tillage with presence or absence of winter cover crops) replicated eight times in 10 m × 100 m plots. The treatments are reduced tillage (transitioning to true zero-tillage in 2012; see Sect. ) with cover cropping (CTCC), reduced tillage with winter fallowing (CTNC), standard tillage with cover crops (STCC), and standard tillage with winter fallowing (STNC) .

Soil collection occurred in October 2019, with sampling strategies designed based on previous findings by , which showed significant management effects on soil hydro-physical properties (porosity, pore size distribution, field capacity) in the 0–5 cm layer but not at 20–25 cm depth. Consequently, sampling focused on 0–5 and 5–10 cm depths.

Seven undisturbed cores were collected from each of the two treatments (CTCC and STNC) at both depths using standardized METER HYPROP cylinders (250 cm3). Treatment selection emphasized the extreme management contrasts, as prior HYPROP measurements showed no significant differences in intermediate treatments (tillage-only or cover crop-only modifications). Core locations were randomly selected on the bed tops within each plot, offset from the bed centerline to avoid the buried subsurface drip irrigation line and crop row.

Complementary bulk samples (0–10 cm) were collected from randomly selected bed-top locations across all four treatments, also offset from the bed centerline, for aggregate stability and density fractionation analyses. A spade shovel was used to dig to approximately 10 cm deep. Then a representative sample of soil was transferred to a rigid plastic container. Upon arrival to the lab, sample containers were left open for 2 weeks to air dry and stored at room temperature.

Wet aggregate stability was determined using a wet sieving apparatus (Eijkelkamp North America Inc.) on air-dried bulk soil samples . Samples were separated through a stack of sieves (8, 4, and 2 mm), yielding four size fractions: > 8, 4–8, 2–4, and < 2 mm. Analysis focused exclusively on macro-aggregates (4–8 and 2–4 mm) because these size classes are most responsive to management-induced changes in soil structure through root and fungal hyphal binding, whereas micro-aggregate formation depends primarily on inherent soil properties . Additionally, micro-aggregates comprised 40 %–70 % more material by volume but were concentrated at depths where treatment effects were minimal, making macro-aggregates more sensitive indicators of surface management impacts. Preliminary testing confirmed that air-dried samples provided optimal discrimination between treatments, as pre-saturated aggregates remained nearly 100 % stable after wet-sieving regardless of treatment.

Two 4 g subsamples were collected from each size fraction, with one subsample used for analysis and the other for moisture content determination. Aggregates from the 4–8 mm fraction were placed in 2 mm mesh sieve cups, while the 2–4 mm fraction was placed in 1 mm mesh sieve cups. Samples underwent wet-sieving in tap water for 3 min, with the stable fraction manually dispersed through the sieve into a separate collection container. All fractions were oven-dried at 100 °C for 48 h before final mass determination.

Wet aggregate stability (WAS) was calculated as the percentage of stable aggregates relative to total aggregate mass, corrected for moisture content and rock fragments: 1WAS=mstablemstable+munstable×100 where mstable is the oven-dry mass of the stable fraction and munstable is the oven-dry mass of the unstable fraction.

Physical property characterization was conducted on intact soil cores through sequential analysis measuring hydraulic conductivity, water retention, and porosity.

Experimental Timeline and Setup: Cores underwent sequential analysis over approximately 14 d: (1) initial saturation (24 h), (2) saturated hydraulic conductivity measurement, (3) continuous drying with water retention measurement (≈12 d), and (4) final moisture determination (24 h). Cores were processed in batches of six containing samples from both treatments and depths to minimize batch effect. Additional methodological details are provided in .

Following physical measurements, soil cores were oven-dried at 105 °C (24 h), gently disaggregated, and passed through a 2 mm sieve. A 5 g subsample from each core was finely ground with an agate mortar and pestle to pass a 150 µm mesh for elemental analysis. Acid reaction testing confirmed negligible inorganic carbonate content, indicating total carbon measurements effectively represented total organic carbon.

Total carbon (TC) and total nitrogen (TN) were determined using dry combustion in an Elemental Analyzer coupled with a Delta V Plus Continuous Flow Isotope Ratio Mass Spectrometer (Costech 4010). Samples (15 ± 0.1 mg) were weighed into tin capsules using a microbalance (Mettler Toledo XP6, precision ± 0.001 mg). Calibration employed certified reference materials USGS 40 and 41a with mean TC values of 40.30 ± 2.10 % (n=7) and 41.59 ± 1.13 % (n=4), and mean TN values of 9.40 ± 0.52 % (n=7) and 9.71 ± 0.26 % (n=4), respectively. Quality control standards were analyzed every 10 samples.

TC and TN stocks were quantified using analytical measurements, soil physical properties, and sample dimensions: 6Si=Ci×ρb×d×(1-fc) where Si represents elemental stock (kg m⁻²) for element i (TC or TN), Ci is measured elemental concentration (kg kg⁻¹), ρb denotes soil bulk density (kg m⁻³), d is sampling interval thickness (m), and fc represents volumetric coarse fraction (>2 mm). The coarse fraction was consistently minimal (<5 %) across all analyzed samples.

Fourier-transform infrared spectroscopy (FTIR) analysis was conducted on surface soil samples (0–5 cm depth) to characterize functional group composition of soil organic matter and assess management-induced changes in organic matter quality.

FTIR spectra were collected using a FTIR spectrometer (Bruker Vertex 70) equipped with a Praying Mantis DRIFTS accessory. Pure potassium bromide (KBr) served as background reference material. Spectra were recorded in absorbance mode from 4000 to 400 cm⁻¹ at 4 cm⁻¹ resolution, with each spectrum representing an average of 300 scans and atmospheric compensation enabled to minimize interference from water vapor and CO₂. Data collection occurred at room temperature under ambient conditions.

Spectral processing was performed using OPUS software (Bruker Cor.). This included baseline correction and smoothing to minimize noise while preserving spectral features. Peak assignments for soil organic matter functional groups were based on established literature values . Key functional groups identified included: aliphatic C-H stretching of methyl groups (2976–2898 cm⁻¹) and methylene groups (2870–2839 cm⁻¹) associated with simple plant materials; aromatic C=C stretching representing complex plant compounds such as lignin (1550–1500 cm⁻¹); and C=O stretching of carbonyl groups in the 1660–1580 cm⁻¹ region, which also includes contributions from amide I bands and aromatic C=C vibrations and is therefore interpreted here as processed (microbially-associated) organic matter rather than as an unambiguous marker of microbial-derived material . These spectral regions exhibit minimal interference from the mineral component, allowing interpretation of organic matter composition.

Density fractionation was performed on air-dried bulk soil samples (0-10 cm depth) to separate soil organic matter into three functionally distinct fractions: free light fraction (FLF), occluded light fraction (OLF), and heavy fraction (HV) closely following , , and . Samples were passed through an 8 mm sieve and homogenized by hand mixing. Approximately 25 g of soil was weighed for fractionation, with a separate 10 g subsample collected for moisture correction (dried at 105 °C for 48 h).

For FLF separation, soil samples were placed in Nalgene conical tubes with 90 mL of sodium polytungstate solution (SPT, density = 1.85 g cm⁻³). Tubes were inverted by hand 10 times and shaken at 150 rpm for 1 h, followed by centrifugation at 3470 rpm for 1 h. The floating FLF was collected by aspiration, filtered through 0.8 µm polycarbonate filter paper, and rinsed five times with 100 mL Milli-Q water before drying at 50 °C for 48 h.

The remaining soil was mixed at 170 rpm for 1 min using a stand mixer, then sonicated in an ice bath to achieve approximately 22 400 J of energy input. After centrifugation at 3470 rpm for 1 h, the OLF was aspirated and purified through 4 repeated washing cycles with 150 mL Milli-Q water, including vortexing (1 min), centrifugation (30–60 min at 3470 rpm), and decanting through 0.8 µm polycarbonate filter paper. The OLF palet formed at the bottom of the conical tube was collected and oven dried at 50 °C for 48 h. The final fraction, HV, underwent identical washing procedures before collection and drying at 105 °C for 48 h.

All fractions were weighed and manually ground for subsequent carbon and nitrogen analysis using the same protocols described for soil cores. Mass recovery and apparent carbon recovery were calculated as the sum of fraction masses (and fraction-weighted carbon) relative to the bulk values. Mass recoveries were consistently within ±4 % (CTCC: 99.7±3.1 %; STNC: 100.5±4.2 %). Apparent carbon recoveries were 89.2±6.4 % (CTCC) and 81.8±7.8 % (STNC); the lowest recovery within STNC (66.4 %, sample STNC18-19, Block 5) coincided with the highest bulk carbon concentration in that treatment, consistent with elevated dissolved organic carbon losses during SPT washing in carbon-rich samples. Sample-level recoveries are reported in Table .

Mineral-associated organic carbon (MAOC) saturation was assessed using the heavy fraction obtained from density fractionation. The analysis employed the clay-silt model and maximum organic carbon estimates established by , which proposes theoretical carbon saturation values of 86 ± 9 mg C g⁻¹ mineral for high-activity minerals and 48 ± 6 mg C g⁻¹ mineral for low-activity minerals.

Soil classification previously conducted at the site identified the soil series as Panoche clay loam (Typic Haplocambids, Aridisol order). Based on USDA soil survey data for this series , the surface horizon (0–18 cm) contains 28 % clay with a cation exchange capacity of 22.2 cmol(+) kg⁻¹, confirming classification as high-activity minerals under the framework (CEC >15 cmol(+) kg⁻¹). Site-specific textural analysis determined clay content ranging from 23 %–32 % and similar proportions for silt (23 %–32 %) , consistent with the soil survey range of 25 %–28 % clay for this series.

MAOC saturation percentage was calculated as: 7MAOCsaturation=CmeasuredCmax⁡ where Cmax⁡ represents theoretical maximum carbon storage capacity (mg C g⁻¹ soil) calculated as the product of mineral (clay+silt) content and the appropriate mineral-specific maximum carbon storage value, and Cmeasured is measured carbon concentration in the MAOC fraction (mg C g⁻¹ soil).

Assessment of data integrity followed a systematic three-phase protocol. Initially, all measurements underwent screening for instrument-specific artifacts according to established validation parameters for each analytical device. Subsequently, mass recovery criteria retained only data exhibiting recovery rates within ±10 % of initial mass values, particularly relevant for density fractionation and aggregate stability determinations.

In the final phase, anomaly detection utilized an adjusted boxplot methodology calibrated for log-normal distribution characteristics of measured variables. Data points falling beyond 1.5× interquartile range (IQR) thresholds were flagged for individual evaluation, with elimination from the final dataset only when definitive instrumental malfunctions or procedural inconsistencies could be identified .

Sample sizes vary by analysis. Aggregate stability (Fig. ) used bulk samples from all eight replicate blocks (n=8 per treatment per size class). Soil-core analyses (Figs. –) yielded n=7 paired blocks after one replicate was excluded for sampling or processing issues. Density fractionation analyses (Figs. –) were performed on bulk samples from six of the eight blocks (n=6 paired blocks; blocks 7 and 8 were not sampled for this analysis); all six samples passed the ±10 % mass-recovery criterion described above.

The CASI experiment is a randomized complete block design with eight blocks arranged along a clay-content gradient. Prior to analysis, all datasets were tested for normality of paired differences using the Shapiro–Wilk test (α=0.05). For two-treatment comparisons (CTCC vs. STNC), paired tests honoring block pairing were used: paired t-tests where the Shapiro–Wilk test on the paired differences indicated normality, and paired Wilcoxon signed-rank tests otherwise. For the four-treatment aggregate-stability comparison (Fig. ), a randomized-block ANOVA was followed by Games–Howell post-hoc tests, which accommodate unequal variances among treatment groups. Boxplots with individual data points (jitter overlay) are used throughout because, at our sample sizes (n=6–7), they convey distributional information that bar plots obscure; statistical inference is based on the paired tests described above, with the specific test reported in each figure caption. The mineral-associated organic carbon saturation analysis additionally propagates the ±9 mg C g⁻¹ mineral uncertainty from the framework through the per-sample saturation calculation using the delta method, with treatment means and 95 % confidence intervals reported. Statistical significance is denoted at p<0.05 (^*), p<0.01 (^**), and p<0.001 (^***). All analyses were performed in R version 4.5.2 ; analysis code and complete dataset are available in the public repository listed in the Data Availability statement.

Aggregate stability (Fig. ) is reported for all four treatments because it provides the empirical motivation for focusing the remaining analyses on the management endmembers (CTCC and STNC). The intermediate treatments (CTNC and STCC) show limited differentiation from STNC, consistent with prior hydro-physical measurements at this site , justifying the endmember focus adopted throughout Sect. –.

Conservation management (CTCC) significantly enhanced wet aggregate stability compared to all other treatments across both analyzed size fractions (Fig. ). In the 2–4 mm aggregate fraction, CTCC exhibited 136 % greater stability (46.9±15.2 %) than STNC (9.6±4.8 %; Games–Howell after RB-ANOVA, p<0.001). Similarly, in the 4–8 mm size fraction, CTCC maintained 52.9±17.8 % stability compared to STNC at 10.9±6.1 % (p<0.001).

Standard tillage with cover crops (STCC) showed significantly higher stability than STNC in both size fractions (2–4 mm: 113 % increase, 20.5±8.6 % vs. 9.6±4.8 %, p=0.008; 4–8 mm: p=0.009). Reduced tillage without cover crops (CTNC) showed intermediate values but did not differ significantly from STNC in the 2–4 mm class (p=0.224), with a significant difference only in the 4–8 mm class (p=0.034). STNC consistently exhibited the lowest aggregate stability values across all measured size classes.

Physical-property measurements on intact soil cores revealed significant differences between reduced tillage with cover crops (CTCC) and standard tillage without cover crops (STNC), concentrated in but not confined to the surface horizon.

Bulk Density: Bulk density under CTCC was significantly lower in the 0–5 cm layer (0.99±0.05 g cm⁻³) than under STNC (1.16±0.02 g cm⁻³; paired t, p=0.024), representing a 15 % reduction (Fig. ). In the 5–10 cm layer, bulk density did not differ significantly between treatments (1.24±0.03 vs. 1.17±0.04 g cm⁻³; p=0.114).

Carbon and nitrogen accumulation patterns varied substantially between management systems and soil depths, with pronounced effects concentrated in the surface horizon.

Carbon Stocks: Total carbon stocks in the 0–5 cm layer under CTCC (1.04±0.07 kg m⁻²) were approximately double those under STNC (0.52±0.01 kg m⁻²; paired t, p<0.001; Fig. ). In the 5–10 cm layer, CTCC (0.72±0.05 kg m⁻²) increased carbon stocks by 36 % compared to STNC (0.53±0.02 kg m⁻²; p=0.002).

Fourier-transform infrared spectroscopy analysis of surface soil samples (0–5 cm) revealed compositional differences in soil organic matter between management systems.

Simple plant-derived organic matter did not differ significantly between CTCC and STNC (26.8±0.8 % vs. 24.7±0.7 %; paired t, p=0.056; Fig. ). The proportion of processed (microbially-associated) organic matter was significantly lower under CTCC than under STNC (48.2±0.2 % vs. 50.4±0.3 %; p<0.001). Complex plant-derived organic matter showed similar proportions between CTCC and STNC (25.0±0.6 % vs. 24.9±0.8 %; p=0.88).

Density fractionation analysis of bulk soil samples (0–10 cm depth) revealed distinct patterns of carbon and nitrogen distribution among functionally different soil organic matter pools.

Mineral-associated organic carbon saturation revealed significant differences between management practices. CTCC exhibited substantially higher carbon saturation than STNC (11.5±1.6 % vs. 7.4±0.8 %; paired t, p=0.007; Fig. ), representing a 55 % relative increase. Saturation was calculated relative to the theoretical maximum carbon storage capacity of 86±9 mg C g⁻¹ mineral for high-activity minerals ; the reported standard errors and 95 % confidence intervals incorporate the ±9 mg C g⁻¹ mineralogical uncertainty propagated through the per-sample saturation calculation using the delta method (CTCC 95 % CI: 7.3 %–15.6 %; STNC 95 % CI: 5.2 %–9.6 %). Both treatments remained well below the theoretical maximum capacity; the implications of this saturation gap are discussed in Sect. .

The dramatic improvement in aggregate stability under reduced tillage with cover crops (CTCC) – exhibiting 136 % greater stability than standard tillage without cover crops (STNC) – represents the cornerstone of soil system transformation and aligns with global patterns from long-term conservation studies. Global meta-analyses consistently demonstrate enhanced aggregate stability as a characteristic response to conservation agriculture, with improvements driven by reduced soil disturbance and enhanced organic matter inputs . The pronounced differences in structural properties between treatments reflect underlying changes in soil biological activity and organic matter dynamics that serve as the foundation for understanding all subsequent improvements.

The substantial improvement in aggregate stability is consistent with synergistic effects of carbon inputs from cover crop residues and reduced physical disturbance through reduced-tillage practices . The four-treatment comparison in Fig.  is consistent with synergistic rather than purely additive effects of combining reduced tillage with cover cropping; this interpretation cannot be extended to variables for which intermediate treatments were not analyzed. Modern understanding of aggregate formation emphasizes the role of fungal networks in creating stable soil structure, with mycorrhizal associations showing enhanced development under integrated conservation management . The enhanced stability observed under CTCC likely reflects development of fungal–bacterial networks that create robust binding agents while simultaneously providing pathways for carbon protection within aggregate structures. Our observation that standard tillage with cover crops (STCC) exhibited greater aggregate stability than reduced tillage without cover crops (CTNC) provides important insights into the relative importance of biological versus physical drivers of soil structure. This pattern aligns with global analyses showing that biological carbon inputs consistently outweigh tillage effects in determining long-term structural improvements . This finding supports the hypothesis that biological processes associated with continuous root activity and residue inputs are primary drivers of aggregate formation and stabilization .

The improved aggregate stability under CTCC creates the physical foundation for all subsequent improvements in soil functioning. Stable aggregates provide protected micro-sites for organic matter accumulation, create favorable conditions for microbial activity, and establish the pore network architecture governing water movement and retention.

Our density fractionation analysis is consistent with a reorganization of carbon protection mechanisms under long-term conservation agriculture. The enrichment of the occluded light fraction under CTCC (44.1 % vs. 35.4 % of total recovered carbon in STNC) indicates that aggregate-mediated physical protection becomes a substantial pathway for carbon storage in this system. We note that the boundary between “chemical” and “physical” protection is not sharp: the OLF carbon resides on organo-mineral surfaces within aggregates, so the shift is more precisely described as a transition from primarily mineral-sorptive protection (HV/MAOC) toward aggregate-mediated protection (OLF), with both mechanisms involving mineral surfaces.

This mechanism shift is broadly consistent with recent global analyses of carbon protection under conservation management. Global cropland analysis from 2000–2022 showed that particulate organic carbon increased 21.8 % while MAOC remained stable across agricultural systems, with North American croplands under conservation policies for more than 20 years showing increases in both POC and MAOC . Our Mediterranean site results are consistent with these global trends, indicating that physically- and chemically-protected carbon coexist as the dominant protection mechanisms when conservation practices are sustained over multi-decade periods.

We interpret the observed mechanism distribution within the temporal-phase framework proposed by , which described initial rapid MAOC formation in years 0–5, continued MAOC accumulation with enhanced microbial processing in years 5–15, and a mature phase beyond 15 years where physical protection becomes increasingly important. We treat this phase progression as a working framework rather than an established trajectory: was a 10-year experiment and did not directly observe the > 15-year phase invoked here, and the climate, soil type, and cropping system differ from ours. Longer-term studies in other systems document declining carbon sequestration rates approaching equilibrium within 15–20 years across diverse climates, providing additional support for the trajectory.

The shift toward aggregate-mediated protection has implications for carbon persistence. While MAOC is often considered more stable due to strong organo-mineral bonds, physically-protected carbon within stable aggregates can achieve similar longevity through reduced exposure to decomposer organisms and limited oxygen availability . Protection mechanism diversity enhances overall carbon stability, with aggregate-mediated protection providing resilience against environmental perturbations that might destabilize MAOC .

FLF carbon concentration did not differ significantly between treatments despite the additional organic-matter inputs under CTCC. This observation is consistent with rapid integration of fresh residues into more stable soil fractions under conservation management, or with elevated decomposition of fresh inputs; the present data do not distinguish between these possibilities, and direct measurement of microbial carbon use efficiency would be required to do so.

The MAOC saturation analysis carries important interpretive caveats for future sequestration potential. CTCC exhibited 55 % higher mineral saturation than STNC (11.5±1.6 % vs. 7.4±0.8 %), with the propagated uncertainty in the theoretical maximum incorporated into the reported standard errors. Both treatments remained well below the theoretical maximum. This saturation gap should not, however, be interpreted as untapped potential under current management. Mediterranean agricultural soils typically show MAOC saturation well below theoretical maxima not because mineral surfaces are unsaturated for chemical reasons but because environmental constraints – low annual precipitation, seasonal aridity, and limited carbon inputs during dry months – set the realized ceiling for organic matter accumulation . If management-related carbon accumulation has approached a quasi-steady state after two decades while substantial mineralogical capacity remains, then mineral surface availability is not the bottleneck. Closing the remaining gap to mineralogical capacity would require fundamentally different interventions (organic amendments, irrigation changes, or deeper-rooted perennial systems) rather than continued application of current conservation practices.

Physical-property changes under CTCC are concentrated in but not confined to the surface horizon. At 0–5 cm, bulk density was 15 % lower and field capacity 12 % greater under CTCC than under STNC, and total porosity was significantly elevated – changes that are consistent with a more developed surface pore network supporting both water storage and movement. These changes are agronomically meaningful in Mediterranean systems where water availability often constrains productivity. Water-retention improvements of 10 %–15 % are typical in Mediterranean climates after 15–20 years of conservation management , and may reduce irrigation requirements and improve drought resilience .

The improved surface pore structure under CTCC is consistent with hierarchical pore networks described for mature conservation systems, in which larger inter-aggregate pores facilitate rapid infiltration during precipitation events while smaller intra-aggregate pores retain water under stress conditions . The depth pattern in porosity is more nuanced than a simple surface-only effect: at 5–10 cm, porosity was significantly lower under CTCC than under STNC, inverting the surface direction. This subsurface contrast is plausibly attributable to the absence under CTCC of the transient deep-tillage loosening that ST achieves during subsoiling operations , leaving the CTCC subsoil less loosened than the ST subsoil immediately after a tillage pass but functionally undisturbed. The vertical stratification – surface accumulation under CTCC, subsurface consolidation under no-till relative to seasonal ST disturbance – is consistent with mature conservation systems where the surface horizon hosts most of the management signal .

Saturated hydraulic conductivity did not differ significantly between treatments at either depth, consistent with the high spatial variability characteristic of Ksat in heterogeneous soils . Mean values were nominally higher under CTCC than STNC at both depths, but the variability among cores is large enough that we do not infer treatment effects on Ksat from these data. Future work employing X-ray computed tomography or other high-resolution imaging could resolve the pore-network architecture that governs water movement in these systems .

The substantial carbon accumulation under CTCC – with surface soil stocks approximately double those in STNC and bulk-soil C concentrations 55 % greater – indicates a meaningful sequestration response to two decades of conservation management. A first-order calculation suggests a retention efficiency of approximately 19 % of cumulative added cover-crop carbon, with the remainder either mineralized or transported below our sampling depth. This estimate uses cumulative cover-crop biomass inputs extrapolated from periodic on-site measurements across the 20-year period and is subject to substantial uncertainty arising from year-to-year variation in cover-crop productivity, composition, and decomposition rate; the value should be read as an order-of-magnitude estimate rather than a precise retention coefficient.

Our carbon accumulation patterns are consistent with the trajectory toward dynamic equilibrium described for conservation agriculture systems managed for 15–20 years . However, our cross-sectional design does not permit direct confirmation of equilibrium status. An important caveat is the evolving management trajectory at this site: as described in Sect. , the CT system permitted shallow cultivation for tomato crops from 1999 to 2011, with true zero-tillage achieved only in 2012. The system therefore experienced approximately 13 years of reduced tillage followed by 7 years of true no-till before sampling. Two implications follow. First, the system may still be responding to the more recent intensification of disturbance reduction, providing an additional reason to interpret the equilibrium framing as a plausible trajectory rather than a confirmed outcome. Second, this progressive adoption pathway is representative of how conservation agriculture adoption typically occurs in practice – through incremental intensification rather than abrupt transitions – so our results capture a realistic adoption scenario. Additional temporal data, including future measurements at this site or analysis of archived soil samples, would be needed to confirm equilibrium status.

Wider C:N ratios under CTCC (9.1 vs. 7.5 at the surface) combined with FTIR evidence of a lower proportion of processed (microbially-associated) organic matter indicate that conservation management preserves more plant-derived characteristics in the soil organic matter pool. Recent evidence suggests that physically-protected organic matter with relatively wide C:N ratios can achieve long-term persistence when embedded within stable aggregate structures .

Nitrogen cycling patterns paralleled carbon dynamics but with distinct fractionation. The OLF carried a disproportionately larger share of recovered N under CTCC than under STNC, while the increase in HV nitrogen was more modest. The redistribution toward aggregate-mediated forms may improve nitrogen synchrony with crop demand and reduce leaching losses .

Our findings from California's Central Valley provide important validation that conservation agriculture principles are effective across diverse climatic conditions, with Mediterranean systems showing particularly strong responses to integrated conservation practices. The Mediterranean climate, characterized by pronounced seasonal moisture patterns and high potential evapotranspiration, creates conditions that enhance conservation agriculture benefits through intensified aggregate formation during wet-dry cycles and increased water use efficiency.

The improvements in this Mediterranean system suggest that these environments may be particularly responsive to conservation management. Global analyses indicate that Mediterranean agricultural regions show some of the highest potential returns on conservation agriculture investments, with water limitation creating strong incentives for practices that enhance water retention and use efficiency . The 12 % improvement in field capacity observed at 0–5 cm represents a localized but potentially valuable enhancement in water-limited systems. While the shallow extent may limit impact on deep-rooted crops during extended dry periods, it could benefit seedling establishment and shallow-rooted crops during critical early growth stages.

The depth stratification we observed – pronounced improvements in surface layers and direction-dependent changes at 5–10 cm – reflects the seasonal nature of Mediterranean precipitation, where enhanced surface infiltration and water storage provide value during the spring growing period. The magnitude of aggregate stability improvements (136 %) exceeds that reported in many temperate-region studies, consistent with the proposition that high clay content combined with pronounced wet–dry cycling creates particularly favorable conditions for aggregate development under conservation management .

Several limitations bound our inferences. Our sampling depth (0–10 cm) is appropriate for detecting management effects but does not capture deeper carbon dynamics; recent work suggests that carbon transport to subsoil layers may represent an important sequestration pathway in conservation systems . The high spatial variability in hydraulic conductivity highlights ongoing challenges in characterizing soil physical properties that exhibit pronounced heterogeneity, motivating future work using X-ray computed tomography or comparable high-resolution imaging . Finally, the physicochemical focus of this study leaves biological mechanisms – microbial community composition, enzymatic activity, microbial carbon use efficiency – unmeasured; these would directly test the mechanistic explanations we have advanced for the observed C and N partitioning.

The improvements in soil health indicators documented in this 20-year study support conservation agriculture as an effective approach for enhancing agricultural sustainability and contributing to climate-change mitigation. The consistency of improvements after two decades is consistent with stable, long-term benefits rather than temporary responses to management changes .

The shift toward aggregate-mediated carbon protection has implications for carbon market protocols, as physically-protected carbon may respond differently to environmental perturbations than mineral-sorptive carbon . Understanding these protection-mechanism differences will be important for accurate carbon accounting and for permanence requirements in carbon credit programs.

The four-treatment aggregate-stability comparison (Fig. ) is consistent with synergistic effects of combining reduced tillage with cover cropping, emphasizing the value of integrated adoption . The substantial benefits seen under standard tillage with cover crops alone (STCC) also support stepwise adoption pathways for systems that cannot transition to integrated conservation management immediately.