Assessing management strategies for carbon storage in Mediterranean soils: double-cropping, no-tillage, and nitrogen fertilization reduction

Assessing management strategies for carbon storage in Mediterranean soils: Double-cropping, no-tillage, and nitrogen fertilization reduction Jesús Fern´ andez-Ortega a,* , Jorge ´ Alvaro-Fuentes b , Antonio Delgado c , Ana María García-L´ opez c , Carlos Cantero-Martínez a a Agricultural and Forest Science and Engineering Dpt. University of Lleida - Agrotecnio CERCA Center, Av. Alcalde Rovira Roure, 191, Lleida 25198, Spain b Soil and Water Dpt., Estaci´ on Experimental de Aula Dei (EEAD), Spanish National Research Council (CSIC), Avd. de Monta˜ nana, 1005, Zaragoza 50059, Spain c Department of Agronomy, University of Seville, Ctra. Utrera km 1, Seville 41013, Spain ARTICLE INFO Keywords: Double-cropping system Legume Maize No-tillage N fertilization Soil organic carbon ABSTRACT In Mediterranean conditions, the historical use of traditional agricultural practices has led to a significant loss of soil organic carbon (SOC) and the associated benefits it provides. Consequently, it becomes imperative to explore effective strategies that promote the preservation and enhancement of SOC. Some promising practices to increase SOC are the use of double-cropping, conservation tillage, and reduced N fertilization. The aim of this study was to evaluate the combined effects of introducing a legume prior to maize, together with different tillage systems and mineral N fertilization rates on SOC and related fractions (particulate organic matter carbon, POM-C; mineralassociated organic matter carbon, Min-C; and permanganate-oxidizable organic carbon, POxC). Additionally, the study aimed to investigate enzymatic activities associated with the carbon cycle. The study compared monocropping maize (MC) versus legume-maize double-cropping (DC) with two tillage systems (conventional tillage, CT; no-tillage, NT), and three mineral N fertilization rates (zero, medium and high). The legumes employed were pea for grain (2019), vetch for green manure (2020), and vetch for forage (2021). The DC increased the SOC level by 10.6 % compared to the use of MC, with POM-C as the main fraction involved in this change. Thus, the employment of DC allowed for the maintenance of SOC levels, while the use of MC resulted in their reduction compared to the levels observed at the beginning of the experiment. NT exhibited higher values of SOC and its fractions POM-C and Min-C. These differences were observed only in the 0–10 cm depth layers. The use of NT enabled the maintenance of SOC compared to the initial studied period, while CT reduced SOC. The treatments with N fertilization achieved higher values of SOC and all the studied fractions compared to the unfertilized treatment. However, at the end of the experiment, it was found that the application of N fertilization, especially at high rates, led to a decrease in SOC. Additionally, it was observed that the employment of DC and NT increased the enzymatic activities of dehydrogenase and β-glucosidase. The results of this study indicate that the utilization of legume-maize DC, as well as the implementation of NT and reduced N fertilization, are useful strategies to maintain SOC levels and improving the biological quality of the soil under Mediterranean irrigated conditions. 1. Introduction Soils are the largest store of terrestrial organic carbon (C), with an accumulation greater than that accumulated by the atmosphere and plants combined, and an estimated figure amount of 1580 Gt C (Jobb´ agy and Jackson, 2000; Cotrufo et al., 2019). Therefore, C sequestration in soils plays a crucial role from an environmental perspective, by sequestering CO 2 from the atmosphere, and from a soil fertility perspective by improving the physical, chemical, and biological properties of the soil (Sainju et al., 2011). The drivers of soil organic carbon (SOC) changes can be grouped into three main categories: 1) climatic variables such as precipitation and temperature; 2) soil conditions such as physical and chemical properties; 3) biotic properties such as the quantity and quality of C inputs to the soil or soil microorganism Abbreviations: CT, conventional tillage; DC, double-cropping; MC, monocropping; Min-C, mineral-associated carbon; NT, no-tillage; NO− 3, nitrate; NH+ 4, ammonium; PNP, p-nitrophenol; POM-C, particulate organic carbon; POxC, permanganate-oxidizable organic C; SOC, soil organic carbon; SOM, soil organic matter; TPF, triphenyl formazan. * Corresponding author. E-mail address: [email protected] (J. Fern´ andez-Ortega). Contents lists available at ScienceDirect Soil & Tillage Research journal homepage: www.elsevier.com/locate/still https://doi.org/10.1016/j.still.2025.106496 Received 18 January 2024; Received in revised form 29 January 2025; Accepted 12 February 2025 Soil & Tillage Research 249 (2025) 106496 Available online 15 February 2025 0167-1987/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). biodiversity (Luo et al., 2017). These drivers create an annually balanced system as the losses of C to the atmosphere through autotrophic and heterotrophic respiration are compensated by the C gain obtained in the soil through photosynthesis. However, the changes of land use towards agricultural systems and the intensification of agriculture are accelerating the losses of SOC to the atmosphere (McLauchlan, 2006; Lal, 2007; García-Palacios et al., 2021). Additionally, the current variability in temperatures and precipitation further complicates the situation. Increases in temperature can contribute to an escalation in SOC losses through accelerated microbial decomposition and root respiration processes (Richter et al., 2007; Stewart et al., 2007; Senthilkumar, 2009). On the other hand, soil drying and wetting processes have also been shown to increase organic matter decomposition by promoting microbial activity (Matteau et al., 2021). This phenomenon has garnered substantial attention from the scientific community in recent decades, evident in the comprehensive models of the climate cycle presented by the Intergovernmental Panel on Climate Change (IPCC, 2014). Within the agricultural domain, a proposed solution for maintaining SOC involves increasing organic matter to the soil through crop residues. A promising technique is the intensification of crops by employing two cultivation cycles per year, also known as doublecropping (Zhao et al., 2018). The effects of these intensified systems on SOC vary depending on the crop species and residue management practices. Double-cropping with legume-cereal combinations and retaining residues on the field has been shown to potentially increase SOC (Stagnari et al., 2017; Jian et al., 2020). However, if crop residues are removed or insufficient fertilization is applied, it may lead to a reduction in SOC (Heggenstaller et al., 2008; Maresma et al., 2019). Nonetheless, there is limited research available indicating the advantage of double-cropping in increasing SOC content. Conservation tillage and optimized nitrogen (N) fertilization are also two interesting practices that have been extensively studied, showing a significant impact on SOC dynamics. Several studies performed in the Mediterranean region have demonstrated that the implementation of conservation tillage may result in a direct increase in SOC, with an average of 0.40–0.50 Mg C ha −1 year −1 observed in the top 30 cm of the soil profile (Plaza-Bonilla et al., 2010; ´ Alvaro-Fuentes et al., 2012; Mazzoncini et al., 2016). However, the SOC sequestration process has limits, and they are closely related to soil properties, climate conditions, and crop complexity. After a change in the tillage system, it takes about 15–50 years to reach an equilibrium level (steady-state) (Follett, 2001; West and Post, 2002; Smith and Chalk, 2020). This equilibrium level also depends on the water regime of the experiment. ´ Alvaro-Fuentes and Paustian (2011) observed that for the same climate conditions, the Century model predicted a SOC sequestration duration of 90 years in irrigated systems, and 70 years in rainfed systems. Such disparities underscore the importance of investigating each specific cropping system within its respective cropping area. N fertilization also has a direct effect on the soil C balance due to its effect on increasing biomass production (Sainju et al., 2002; Bolinder et al., 2020). However, excessive N fertilization may also lead to increased organic matter mineralization and the release of C into the atmosphere, as well as potential N loss through leaching (Mazzoncini et al., 2011). The biological quality of soils is intricately tied to their capacity for organic matter mineralization and, consequently, SOC sequestration. Enzymatic activities, commonly utilized in studies, serve as valuable indicators of soil biological quality, displaying correlations with different SOC fractions and rates of organic matter mineralization. (Moeskops et al., 2010; Zhang et al., 2021). Dehydrogenase activity, being exclusively measured within the cells of living organisms, serves as a real-time indicator of soil biological activity and respiration (Li et al., 2019). Conversely, β-glucosidase activity is an extracellular enzyme with a central role in cellulose degradation. Since its substrate is rarely limited, fluctuations in this enzymatic activity are ascribed to alterations in soil physical and chemical properties (Eivazi and Tabatabai, 1988; Tan et al., 2021). Thus, studying enzyme activities in conjunction with SOC can give a deeper understanding of the impacts of agricultural management on SOC changes. It is imperative to investigate cultivation techniques that not only maintain SOC but also sustain crop yields, as highlighted by several authors (Blanco-Canqui, 2022; van der Pol et al., 2022). Due to the multitude of factors involved in the current process of SOC loss, conducting studies across diverse geographical areas becomes necessary (Navarro-Pedre˜ no et al., 2021). These studies should encompass comprehensive crop management, including different cultivation systems, soil practices, and fertilizer rates. Therefore, the objectives of this study were: i) to assess the impact of legume-maize double-cropping, in conjunction with different tillage systems and mineral N fertilization rates, on SOC content; ii) to analyze how the main SOC fractions are affected by these changes in cropping management; and iii) to determine the activities of dehydrogenase and β-glucosidase enzymes and their relationships with the observed SOC changes. The initial hypothesis suggests that the use of double-cropping, conservation tillage, and reduced nitrogen rates would result in the highest carbon accumulation, leading to the most significant increases in SOC levels. Moreover, the soil conditions created by these combined management strategies would foster an environment conducive to higher enzyme activities, thereby reflecting improved soil quality. 2. Material and methods 2.1. Experimental design and management practices The study was conducted in northeaster Spain, in the Agramunt municipality (41◦48 ′ N, 1◦07 ′ E, 330 m asl). The region is typical of a semiarid Mediterranean dryland with a continental trend. In the last 30 years, the average annual precipitation has been 442 mm, the average annual temperature has been 14.6 ◦C, and the annual potential evapotranspiration (PET) is 855 mm. The average annual temperature during the three-year duration of the experiment was 15.9 ⁰C, whereas it had been 14.3 ⁰C during the 3 years prior to the experiment (Fig. 1a). The average soil moisture (0–10 cm depth) for both the experimental period and the period before the experiment was 25.3 and 18.3 % respectively (Fig. 1b). According to the Soil Survey Staff (2014), the soil is classified as Typic Xerofluvents. The main physiochemical properties are presented in Table 2. The experimental field was established in 1996 to compare three rates of mineral N (0, 60 and 120 kg N ha −1 ) and three tillage systems (conventional tillage, CT; minimum tillage, MT; no-tillage, NT) under rainfed barley monocropping conditions (Ang´ as et al., 2006) (Table 1). In 2015, the experiment was transformed to irrigated condition using Fig. 1. Daily air temperature (a) and soil volumetric moisture (b) for period 0 (2015–2017) and period 1 (2019–2021). J. Fern´ andez-Ortega et al. Soil & Tillage Research 249 (2025) 106496 2 solid set sprinklers spaced at 18 ×18 m, and the crop was changed to maize (Zea mays L.) monocropping (Pareja-S´ anchez et al., 2017). The tillage treatments were maintained, and mineral N fertilization rates were adapted to maize crop (0, 200, 400 kg N ha −1 ) with the same experimental layout as the previous rainfed experiment. In 2018, to enhance the study of crop diversification and its interaction with tillage and N fertilization, the plots were divided into a split-plot design measuring 3 m wide and 48 m long. This allowed for a comparison between two cropping systems (Cs): monocropping maize (MC) and double-cropping legume-maize (DC). Soil tillage treatments (Till) were the same as those used previously, although only the two most different treatments (CT and NT) were used for this study. The N fertilization rates (Fert) were zero (0 kg N ha −1 ), medium (200 kg N ha −1 ), and high (400 kg N ha −1 ) for MC while assuming the biological fixation of the legume, the rates were adjusted to zero (0 kg N ha −1 ), medium (150 kg N ha −1 ), and high (300 kg N ha −1 ) for DC. Consequently, the crop diversification treatment spanned 3 years, whereas the tillage and N fertilization treatments extended over 25 years by the conclusion of the current experiment. For MC maize, a long-cycle maize cultivar (FAO 700, Pioneer’s P1570 hybrid) was used. For DC a short-cycle maize cultivar (FAO 400, Pioneer’s P0312 hybrid) was planted as summer crop and a legume as winter crop. The legumes were: pea used for grain (Pisum sativum L., var. Furious) during the 2018–2019 season; and vetch (Vicia sativa L., var. Prontivesa) employed as green manure during 2019–2020 and forage during 2020–2021. Throughout the three-year study period, MC maize was sown in April; DC maize in early June; and the legume from December to January. The sowing rate in maize (MC and DC) was 90,000 seeds ha −1 with a separation between lines of 73 cm. In the case of legumes, a density of 100 plants m −2 was used for pea and 267 plants m −2 for vetch. The CT treatment consisted of subsoiler (35 cm depth) followed by one pass of rototiller (15 cm depth) and one pass of roller before planting, with almost 100 % of the crop residues incorporated into the soil. NT plots were sprayed with herbicide, 1.5 L ha −1 of 36 % glyphosate [N-(phosphonomethyl)-glycine] without disturbing the soil. Planting was done with a pneumatic row direct seeding machine equipped with double disc furrow openers (model Prosem K, Sol`a, Calaf, Spain). N fertilizer was manually applied, and exclusively to the maize crop. In MC, pre-sowing urea (46 % N) fertilization took place in April-May. The urea was broadcasted on the soil surface and incorporated through tillage in CT, while it was left on the ground in NT. The presowing fertilization rate was 50 and 100 kg N ha −1 for the medium and high rates, respectively. Furthermore, both in MC and DC, two topdressing fertilizations were performed in stages V3-V5 (May in MC, and late June in DC) and V7-V8 (June in MC, and July in DC). These topdressing applications involved ammonium nitrate (34.5 % N) at 75 and 150 kg N ha −1 for the medium and high rates, respectively. At the onset of each growing season, mineral P and K fertilizers were applied to fulfill the nutritional demands of maize and legumes, in accordance with the standard yields observed in the region. Irrigation was conducted from March to October based on the crop’s water requirements. The irrigation schedule was determined by the crop with the highest water demand, which was estimated weekly by subtracting the effective precipitation (75 % of the total weekly precipitation) from the crop evapotranspiration (ETc) (Dastane, 1978). The ETc was determined using the FAO Penman-Monteith methodology with meteorological data obtained from a nearby weather station in the vicinity of the field experiment. This data was then multiplied by the crop coefficient (Kc) specific to the crop, derived as a function of thermal time (Allen et al., 1998). Table 1 Management history of the experimental field. System: Rainfed Starting year 1996   Crop: Barley Treatments Nitrogen (Kg N ha −1 ) Zero - 0  Medium - 60  High - 120  Tillage Conventional tillage  No tillage  System: Sprinkler irrigation    Starting year 2015     Crop: Maize Treatments Nitrogen (Kg N ha −1 ) Zero - 0   Medium - 200   High - 400   Tillage Conventional tillage   No tillage   2019 Treatments Crop: Maize Legume-Maze Nitrogen (Kg N ha −1 ) Zero 0 0 Medium 200 150 High 400 300 Tillage Conventional tillage   No tillage   Cropping diversification Monnocropping maize   Legume-Maize Double-cropping   Table 2 Soil properties of the Ap horizon (0–28 cm depth) in 1996. Initial soil organic carbon content (SOC i ) (1996) and soil organic carbon content (SOC) (0–30 cm) in three tillage systems (conventional tillage, CT; no-tillage, NT) in 2017. Soil properties Soil classification*Typic Xerofluvent pH (H 2 O, 1:2.5) 8.5 EC 1:5 (dS m −1 ) 0.15 P Olsen (mg kg −1 ) 35 K Amm. Ac. (mg kg −1 ) 194 Water retention (g g −1 ) −33 kPa 0.16 −1500 kPa 0.05 Soil Texture (g kg −1 ) Sand (2–0.05 mm) 308 Silt (0.05–0.002 mm) 573 Clay (<0.002 mm) 119 SOC (g kg −1 ) 1996 2017 7.6 CT 8.6 NT 12.2 * According to the USDA classification (Soil Survey Staff, 2014) J. Fern´ andez-Ortega et al. Soil & Tillage Research 249 (2025) 106496 3 2.2. Soil sampling and analysis The initial soil sampling was conducted in November 2018 prior to the start of the experiment. The final soil sampling was conducted in November 2021, following the harvest of maize. Soil samples were taken in two observations per plot at: 0–5, 5–10, 10–20, 20–30, 30–40 cm of depth. Total soil organic carbon (SOC), permanganate-oxidizable organic C (POxC), particulate organic matter carbon (POM-C) and mineral-associated organic carbon (Min-C) were analysed. These specific fractions were chosen due to their varying sensitivity to changes resulting from different agricultural management practices (´ Alvaro-Fuentes et al., 2014). SOC determination was performed following the methodology of Walkley and Black (1934), using a 1 g soil subsample, previously dried and sieved at 2 mm. POM-C and Min-C fractions were analysed according to Cambardella and Elliott (1992) with a modification developed for our conditions by Pareja S´ anchez et al. (2020). Briefly, twenty-gram subsamples of soil from each depth and plot were dispersed in 100 mL of 5 g L −1 sodium hexametaphosphate for 15 h on a reciprocal shaker. Then the samples were passed through a 50 μ m sieve to separate the POM and Min-C. The material passing through the sieve (Min-C) was collected in aluminium pans and oven dried at 50 ◦C. The wet oxidation method of Walkley and Black (1934) was then used to measure the C concentration in the Min-C fraction. The POM-C content was determined as the difference between total SOC content and Min-C content. POxC was determined according to Weil et al. (2003). SOC contents were determined based on mass per unit area, calculated by multiplying the carbon concentration values obtained through the oxidation method by the corresponding soil bulk density values. Additionally, SOC stock (kg C ha −1 ) was adjusted for equivalent soil mass according to the procedure outlined by Ellert and Bettany (1995) for the 0–40 cm soil depth interval. The annual SOC sequestration rate (ΔSOC rate) (kg C ha −1 yr −1 ) (0–40 cm soil depth) was computed for each treatment from 2018 to 2021. The objective concerning this parameter was to assess the continued use of irrigated maize versus the practice of legumes-maize double-cropping. Soil sampling for enzymatic activities was performed in two observations per plot at 0–5, 5–10, 10–20, 20–30 cm soil depths, exclusively in the CT and NT plots. Soil of two observations were mixed and passed through a 2 mm sieve to obtain a representative and unique sample at different depths. Dehydrogenase activity was determined from 3 g of fresh soil following the method of Casida et al. (1964) based on the determination of triphenyl formazan (TPF) produced from 2,3,5-triphenyl tetrazolium chloride after 24 h incubation at 37 ◦C in the dark. β-glucosidase activity was analysed from 1 g of fresh soil following the method of Eivazi and Tabatabai (1988) based on the determination of p-nitrophenol (pNP) after incubation with p-nitrophenyl-β -D-glucoside for 1 h. 2.3. Carbon inputs Crop residue samples were taken just after harvesting. The method of sampling varied depending on the type of crop. For both MC and DC maize, plant samples from 2-m-long central rows were taken in three observations per plot. For pea, 0.5-m-long of two adjacent rows were sampled at two observations per plot. For vetch 0.36 m 2 of plants were cut at the soil surface level in two areas of each plot. Only the above-ground biomass of the crop was considered for the C inputs, as the grain was harvested for sale and the roots were not sampled. Similarly, the vetch crop in 2021 was not considered in the calculations as it was exported as forage. The biomass of all crops was dried in an oven at 60 ⁰C for 48 h, threshed and weighed excluding the grain, hereafter referred as crop residues. The C content was determined by dry combustion (model Truspec CN, LECO, St Joseph, MI, USA). C inputs were calculated by multiplying the biomass of the agricultural residues by their C content. 2.4. Statistical analysis Data were checked for normality, homoscedasticity and serial independence by Shapiro-Wilk, Bartlett, and Durbin-Watson test respectively. Outliers were checked using the Grubbs test with a statistical confidence level of 95 %. Any data not passing either test required additional transformation. For C inputs and enzymatic activities data analysis, a repeated measures analysis of variance (ANOVA) was carried out with the cropping system, tillage and N fertilization, year, and their interactions as effects. For SOC and its fractions, and for SOC sequestration rate, ANOVA was conducted with the treatments of cropping system, tillage and N fertilization, depth, and their interactions as effects. Statistical analyses were performed with the statistical package JMP pro 16 (SAS Institute Inc., 2021) and Statgraphics Centurion 18 (Statgraphics Technologies Inc., 2018). 3. Results 3.1. Carbon inputs and SOC The results of this experiment correspond to the period 2019–2021 (P1). To explain the observed changes in SOC during this time, data on C inputs from the previous study period (P0, 2015–2017) by Pareja S´ anchez et al. (2020) are also presented (Fig. 2, P0). During P1, the three treatments studied had a significant impact on C inputs (Table 3). Among the cropping systems, DC produced the highest C input values, particularly when combined with NT and medium or high N fertilization rates. The average C inputs for these combinations ranged between 7086 and 8257 kg C ha⁻¹ (Fig. 2). In contrast, the lowest C inputs were consistently observed in the MC-CT combination, regardless of the N fertilization rate, with an average of 3323 kg C ha⁻¹ across all rates. When comparing C inputs across periods, the inputs from P0 were similar to those of the MC treatment during P1, with averages of 3100 kg C ha⁻¹ (P0) and 3650 kg C ha⁻¹ (P1), respectively. By the conclusion of the experiment in November 2021, SOC levels were significantly influenced by all three treatments (Table 3). The DC system showed the highest SOC concentration (9.4 g C kg⁻¹ soil). Regarding tillage management, NT resulted in the highest SOC levels (9.8 g C kg⁻¹ soil). For N fertilization, significant differences were observed between the fertilized treatments and the unfertilized control, with a mean SOC concentration of 8.7 g C kg⁻¹ soil across fertilized treatments. However, no significant differences were found between medium and high N fertilization rates. Fig. 2. Tillage (CT, conventional tillage; NT, no-tillage) and rate of N fertilization (Zero, Med and High: 0, 200, and 400 kg N ha −1 for MC; 0, 150, and 300 kg N ha −1 for DC), effects on C inputs (period 0); and cropping system (MC, monocropping system; DC double-cropping system), tillage and rate of N fertilization effects on C inputs (period 1). For period 0, there are no significant differences among treatments; for period 1, the various lowercase letters indicate significant differences among treatments at p<0.05. The vertical bars indicate the standard error. J. Fern´ andez-Ortega et al. Soil & Tillage Research 249 (2025) 106496 4 When comparing SOC levels at the beginning and the end of the experiment, the ΔSOCrate was also significantly influenced by all three treatments studied (Table 4). Under the DC system, the C stock remained stable compared to 2018, while the MC system experienced a decline in C stock at a rate of 178 kg C ha⁻¹ year⁻¹ . In terms of tillage management, NT effectively preserved soil C stock, whereas CT led to a reduction of 199 kg C ha⁻¹ year⁻¹ . Regarding nitrogen fertilization, the application of N, especially at high rates, caused a reduction in C stock, with decreases of 90 and 157 kg C ha⁻¹ year⁻¹ for medium and high N rates, respectively. In contrast, the unfertilized treatment maintained C stock levels. SOC fractions revealed significant changes associated with the cropping system. Specifically, the use of DC resulted in a 19.5 % increase in POM-C and a 12.7 % increase in POxC compared to MC. The tillage treatment also impacted the POM-C and Min-C fractions, with NT showing POM-C values 44.4 % higher than CT. N fertilization influenced all studied SOC fractions. As with total SOC content, significant differences were observed only between fertilized treatments and the unfertilized control, with no notable differences between medium and high fertilization rates. On average, fertilized treatments showed increases of 13.8 % in POM-C, 11.0 % in Min-C, and 12.5 % in POxC compared to the unfertilized treatment (Table 4). A significant interaction between the tillage system and sampling depth was also observed for both total SOC and its fractions (Table 4). In the topsoil layer (0–5 cm), NT had the highest SOC concentration (18.5 g C kg⁻¹ in NT vs. 11.0 g C kg⁻¹ in CT) (Fig. 3a). At 5–10 cm depth, NT also maintained significantly higher values than CT. However, at deeper layers (10–40 cm), no significant differences were found between tillage treatments, with an average SOC concentration of 4.5 g C kg⁻¹ . For POM-C, the highest values were observed in the surface layer (0–10 cm) under NT, with concentrations of 11.5 and 6.4 g C kg⁻¹ at 0–5 cm and 5–10 cm depths, respectively. No significant differences were found between tillage treatments at intermediate or deeper layers (Fig. 3b). Min-C values were also higher under NT in the 0–10 cm range, while similar values were recorded at greater depths across all tillage treatments (Fig. 3c). For POxC, significant differences between treatments were observed only in the most superficial layer (0–5 cm), where NT recorded a mean value of 760.5 mg C kg⁻¹ , significantly higher than CT at 572 mg C kg⁻¹ (Fig. 3d). 3.2. Soil enzymatic activities The effect of cropping on dehydrogenase activity was found to depend on tillage treatments activity as revealed by the significant interaction between factors. Dehydrogenase activity was notably higher in the NT system, especially when combined with DC, reaching 5.5 μ g TPF g −1 dry soil h −1 (Fig. 4a). The significant differences found in β-glucosidase activity were attributed to individual factors of cropping and tillage systems (Table 3). β-Glucosidase activity in the DC system was 23 % higher compared to MC (Fig. 4b). Additionally, NT resulted in a 53 % increase in β-glucosidase activity (Fig. 4c). 4. Discussion 4.1. Legume-maize double-cropping effects The findings of this study demonstrate that replacing winter fallow in monocropping maize systems with a legume significantly increases SOC levels. It is well-established that higher C inputs consistently act as the primary driver of SOC accumulation across diverse environments (Kong et al., 2005; Liu et al., 2014). In this experiment, the highest C inputs were observed in the DC system, primarily due to the presence of two crop phases per year and the improvements in DC maize yields provided by the preceding legume phase (Talukder et al., 2022; Fern´ andez-Ortega et al., 2023b). This finding was further supported by a statistically significant correlation between SOC levels and soil inputs, modeled through a two-degree polynomial regression (p <0.001, r² =0.22). The analysis of C fractions revealed that the primary fraction affected by the use of DC was POM-C. This fraction is composed of Table 3 Analysis of variance (p-values) of carbon inputs (C-input), soil dehydrogenase activity (DHA) and soil β-Glucosidase activity (β-Glu) as affected by cropping system, tillage, rate of N fertilization, year and their interactions. Source of variation C inputs DHA β-Glu Cropping system (Cs) <0.001 NS <0.01 Tillage (Till) <0.001 NS <0.001 N fertilization (Fert) <0.001 NS NS Cs ⋅ Till NS NS NS Cs ⋅ Fert NS <0.05 NS Till ⋅ Fert NS <0.001 <0.001 Cs ⋅ Till ⋅ Fert <0.05 NS NS Year <0.001 NS NS Year ⋅ Cs <0.001 NS NS Year ⋅ Till NS <0.001 NS Year ⋅ Fert <0.001 NS NS Year ⋅ Cs ⋅ Till NS NS NS Year ⋅ Cs ⋅ Fert <0.05 NS NS Year ⋅ Till ⋅ Fert NS NS NS Year ⋅ Cs ⋅ Till ⋅ Fert NS NS NS NS, not significant Table 4 Analysis of variance at the end of the experiment (November 2021) of soil organic carbon (SOC), particulate organic matter carbon (POM-C), mineralassociated carbon (Min-C), permanganate-oxidizable organic C (POxC) concentration and soil organic carbon sequestration rate (ΔSOCrate) as affected by cropping system (MC, monocropping system; DC, double-cropping system), tillage (CT, conventional tillage; NT, no-tillage), rate of N fertilization (Zero, Med and High: 0, 200, and 400 kg N ha −1 for MC; 0, 150, and 300 kg N ha −1 for DC), depth, and their interactions. Negative values of ΔSOCrate imply a decrease in soil carbon stock compared to the year 2018. Source of variation SOC POM-C Min-C POxC ΔSOCrate  g C kg soil −1 mg C kg soil −1 kg C ha −1 yr −1   MC 8.5 b 4.1 b 4.4 426.8 b −178.1 b DC 9.4 a 4.9 a 4.5 480.0 a 19.1 a CT 7.7 b 3.6 b 4.1 b 448.6 −199.4 b NT 9.8 a 5.2 a 4.6 a 463.0 6.9 a Zero 8.1 b 4.0 b 4.1 b 418.4 b −13.3 a Med 8.9 a 4.4 a 4.5 a 467.3 a −89.5 ab High 9.1 a 4.5 a 4.6 a 474.5 a −156.9 b ANOVA      Cropping system (Cs) <0.01 <0.001 NS <0.05 <0.001 Tillage (Till) <0.001 <0.001 <0.001 NS <0.001 N fertilization (Fert) <0.05 NS <0.05 NS <0.05 Cs ⋅ Till NS NS NS NS NS Cs ⋅ Fert NS NS NS NS NS Till ⋅ Fert NS NS NS NS NS Cs ⋅ Till ⋅ Fert NS NS NS NS NS Depth <0.001 <0.001 <0.001 <0.001 <0.001 Depth ⋅ Cs NS NS NS NS NS Depth ⋅ Till <0.001 <0.001 <0.001 <0.05 NS Depth ⋅ Fert NS NS NS NS NS Depth ⋅ Cs ⋅ Till NS NS NS NS NS Depth ⋅ Cs ⋅ Fert NS NS NS NS NS Depth ⋅ Till ⋅ Fert NS NS NS NS NS Depth⋅ Cs ⋅ Till ⋅ Fert NS NS NS NS NS NS, not significant. Different lower case letters indicate significant differences among treatments at p<0.05. J. Fern´ andez-Ortega et al. Soil & Tillage Research 249 (2025) 106496 5 undecomposed or partially decomposed materials, including microbial biomass, plant residues, and root remnants (Six et al., 2001; Von Lützow et al., 2008). It exhibits the most rapid changes in response to variations in C inputs, typically within weeks to years (Culman et al., 2012; Plaza-Bonilla et al., 2014; Wooliver and Jagadamma, 2023). Moreover, it has been identified as a reliable predictor of changes in the content and quality of soil organic matter, as well as a significant source of potentially mineralizable organic matter in the soil (Semenov et al., 2019). In this study, POM-C levels were significantly higher in the DC system, further reinforcing the idea that changes in SOC are primarily driven by differences in C inputs. In contrast, the use of DC did not lead to significant differences compared to MC in the Min-C fraction. Min-C consists of single molecules or microscopic fragments of organic material leached from plant material or transformed by soil biota (Lavallee et al., 2020; Rocci et al., 2020). Due to the long mean residence time of crop residues, noticeable increases in this fraction typically take decades to centuries (Cambardella and Elliott, 1992; Srivastava et al., 2016). Min-C is also protected from decomposition by associations with soil minerals, such as chemical bonds with mineral surfaces and occlusion within micropores or small aggregates (<50–63 µm), limiting its accessibility to decomposers and enzymes. As a result, crop management changes have a slow impact on Min-C levels (Totsche et al., 2018; Cotrufo et al., 2019). In this study, the three-year DC implementation was insufficient to produce significant differences in Min-C compared to MC systems. The use of DC increased the content of POxC compared to MC. POxC represents the early decomposition products of plant and faunal biomass, root exudates, and microbial biomass (Bolan et al., 2011; Morrow et al., 2016). Its rapid formation makes POxC the most active SOC component (Zhang et al., 2021), often used as an effective indicator of short-term changes in SOC fractions (Plaza-Bonilla et al., 2014; Gabarr´ on-Galeote et al., 2015). The incorporation of easily degradable legumes influences the degradation of organic matter, converting stable C pools into more labile C forms (Li et al., 2019). Furthermore, the DC system exhibited higher dehydrogenase and β-glucosidase activities. Dehydrogenase activity, which exists only in intact cells, reflects the level of active microbial activity at a given time (Veum et al., 2014). It is associated with microorganisms responsible for breaking down organic materials (Ross, 1971; Bolton et al., 1985; Piotrowska-Długosz et al., 2022), leading to rapid increases in soil POxC (Leno et al., 2021) and explaining the higher POxC values observed under DC. Similarly, β-glucosidase activity increases with the addition of easily degradable legume residues (Eivazi and Tabatabai, 1990; Navas et al., 2011). This enzyme plays a key role in the final breakdown of plant debris rich in cellulose (Turner et al., 2002). Higher β-glucosidase activity has been linked to increased levels of Min-C and POxC in various studies (Ajwa and Tabatabai, 1994; Whitbread et al., 2000; Haynes, 2005). Combined, these enzymatic activities suggest that legumes in the Fig. 3. Soil organic carbon (SOC) (a), particulate organic matter carbon (POM-C) (b), mineral-associated carbon (Min-C) (c) and permanganate-oxidizable organic C (POxC) (d) as affected by tillage (CT, conventional tillage; NT, no-tillage) at different soil depths at the end of the study (2021). Within a soil layer Different lower case letters indicate significant differences among treatments at p<0.05. J. Fern´ andez-Ortega et al. Soil & Tillage Research 249 (2025) 106496 6 DC system enhance microbial activity and, consequently, improve C dynamics and fixation in the soil (Visser and Parkinson, 1992; de la Paz Jimenez et al., 2002). The continuous use of MC under irrigated conditions led to a loss of SOC compared to the start of the experiment in 2019. Although the C inputs from MC during this period (P1, 2019–2021) were comparable to those reported in the previous study by Pareja-S´ anchez et al. (2020) (P0, 2015–2017), conducted in the same experimental field under identical tillage and N treatments, they were insufficient to maintain SOC levels. It has been demonstrated that sustained irrigation can increase enzymatic activity and accelerate the mineralization of organic matter, leading to SOC reductions, particularly under frequent cycles of soil wetting and drying (Nunes et al., 2007; Matteau et al., 2021). Furthermore, numerous global studies have consistently identified a strong correlation between declining SOC and increases in temperature and humidity (Senthilkumar, 2009; Morug´ an-Coronado et al., 2020; Mariappan et al., 2022). This decline is primarily driven by accelerated decomposition rates (Davidson et al., 2000; Conant et al., 2008) and increased residue mineralization (Rustad et al., 2001; García-Palacios et al., 2021). In this particular study, the annual mean temperature and maximum temperature were 1.6 ◦C and 3.5 ◦C higher, respectively, compared to the previous period. Additionally, soil moisture was, on average, 7 % higher. Thus, the continued use of intensive irrigation systems, combined with increased temperature and soil moisture, has been identified as the primary factors contributing to the observed reduction in SOC within the MC system. In contrast, using legumes before maize cultivation helped maintain SOC levels. Research has shown that incorporating legumes into rotations can help preserve SOC, especially when residues added to the soil have a low C/N ratio (Paustian et al., 2019; van der Pol et al., 2022). DC not only increases C inputs but also enhances soil aggregate formation (Talukder et al., 2023). This improved structure physically protects C inputs from microbial decomposition (Negassa et al., 2015; Virk et al., 2022). Additionally, legumes alter the soil microbiome’s composition and strategies, enabling efficient decomposition and utilization of plant-derived C sources rather than relying solely on existing organic C (Malik et al., 2020). These effects promote efficient transformations among SOC fractions, leading to stable organic C sequestration through microbial processes (Li et al., 2023). However, despite the higher decomposition rates observed in this study, the increased C inputs under DC were insufficient to augment C stocks compared to the experiment’s beginning, suggesting that the DC system has reached a steady-state (Powlson et al., 2011; Nicoloso et al., 2016; van der Pol et al., 2022). 4.2. Tillage systems effects In line with the initially proposed hypothesis, the NT system showed an increase in SOC and its fractions, POM-C and Min-C, compared to CT. Similar to the cropping system treatment, the higher levels of POM-C in the NT system are primarily attributed to the increased C inputs observed in NT, relative to CT. Numerous studies under comparable conditions have shown that NT systems improve soil physical properties, including the formation of soil macroaggregates, enhanced soil structure, and increased soil water content (Lampurlan´ es and Cantero-Martínez, 2003; Pareja-S´ anchez et al., 2017; Talukder et al., 2022), which in turn supports higher biomass yields. Furthermore, the increase in POM-C is particularly associated with the NT system, mainly due to the better protection provided by soil aggregates in this tillage system and the reduced burial of crop residues compared to CT (Zuber and Villamil, 2016; Kan et al., 2020; Vilakazi et al., 2022). These differences are especially noticeable in the upper soil layers, where the highest concentration of residues accumulates and where this fraction is more sensitive to tillage operations (Wander and Bidart, 2000; Li et al., 2022). In the case of Min-C, there have been limited studies on tillage that Fig. 4. Dehydrogenase activity as effects of the interaction of cropping system (MC, monocropping system; DC, double-cropping system) and tillage (CT, conventional tillage; NT, no-tillage) (a), and β-glucosidase activity as affected by the cropping system (b) and tillage (c). The various lowercase letters indicate significant differences among treatments at p <0.05. The vertical bars indicate the standard deviation. J. Fern´ andez-Ortega et al. Soil & Tillage Research 249 (2025) 106496 7 reported significant results (Poeplau and Don, 2015; Wooliver and Jagadamma, 2023). This is due to the recalcitrant nature of this fraction of C, as well as the typically larger pool size in mineral soils, which leads to very slow changes (Rocci et al., 2021). In our study, although the present experiment lasted 3 years, the 25 years of maintaining the same tillage practices in the total duration of the long-term experiment have allowed us to observe the highest levels of Min-C in the NT. The main justifications for the increase in Min-C are the improved conditions for the establishment and proliferation of microbiota responsible for the transformation and stabilization of SOC provided by NT (Lavallee et al., 2020; Nunes et al., 2020). One of the factors that strongly influences enzyme activity is soil moisture (Adetunji et al., 2017). Specifically, in this trial, significantly higher soil moisture levels were found with the use of NT (Fern´ andez-Ortega et al., 2023a). The combination of higher moisture levels and reduced soil disturbance in NT led to increased dehydrogenase and β-glucosidase activities, which in turn implied greater SOC stabilization (´ Alvaro-Fuentes et al., 2013; Chen et al., 2019). However, the differences found regarding Min-C were limited to the surface layers of the soil. Min-C is related with mineral associations, including chemical bonds between soil organic matter (SOM) and mineral surfaces and occlusion within micropores or small aggregates (<50–63 µm) (Lavallee et al., 2020). It has been proven that NT promotes the formation of micropores and microaggregates, particularly in the 0–10 cm layer (Gregorich et al., 1993; Mondal and Chakraborty, 2022). On the other hand, CT promotes the formation of macroaggregates (Talukder et al., 2023), justifying the lower values of Min-C. In deeper soil layers, differences in aggregation are less pronounced. It is well established that the primary mechanism for stabilizing carbon in the Min-C fraction at these depths is chemical rather than physical. As a result, the differences in this fraction between tillage systems tend to diminish with increasing soil depth (Tivet et al., 2013). Regarding POxC, differences were only observed in the top 5 cm of soil due to its greater mobility (Shen et al., 2021). The higher microbial populations and increased respiration activity near the soil surface accelerate the oxidation of compounds comprising POxC or their transformation into more stable and protected SOC, compared to deeper soil layers (Wang et al., 2017). These processes are particularly observed when there is an abundance of organic matter inputs (Plaza-Bonilla et al., 2014; Jagadamma et al., 2019). When analyzing the effects of tillage at different depths on total SOC content, it was found that NT systems only increased SOC in the top 10 cm of soil. Similarly, experiments conducted in the Mediterranean region showed that after 15 years of experimentation, NT did not significantly increase SOC throughout the entire soil profile, but only in the top 10 cm. In contrast, CT exhibited the highest SOC levels when considering depths up to 40 cm (Huggins et al., 2007; ´ Alvaro-Fuentes et al., 2008; Meurer et al., 2018). In our case, at depths ranging from 10–40 cm, NT systems showed similar SOC values to CT. Studies such as those by Blanco-Canqui and Lal (2008) have demonstrated that although C mobility is higher in CT systems than in NT, long-term maintenance of NT promotes the transfer of carbon to deeper soil layers, thus eliminating the differences between NT and CT over time (Huggins et al., 2007; Meurer et al., 2018; Zhang et al., 2018). The analysis of SOC evolution with the maintenance of tillage systems revealed that while CT resulted in a decrease in SOC, NT allowed for the maintenance of achieved C levels. In CT systems, moldboard plowing accelerates the decomposition of SOC and loss of C to the atmosphere as CO 2 . The mixing of residues and soil through plowing enhances physical contact between soil microorganisms and crop residues, creating more favorable soil microclimatic conditions for residue decomposition (Bruce et al., 1999; Nunes et al., 2020). In contrast, NT practices have the potential to increase SOC sequestration by reducing SOC degradation, lowering SOC mineralization rates, and increasing C inputs (Aguilera et al., 2013; Blanco-Moure et al., 2013; Mazzoncini et al., 2016). It is generally accepted that after 14–15 years of NT practices, a state of SOC equilibrium is achieved, where no significant gains or losses are observed in the studied horizons (´ Alvaro-Fuentes et al., 2008; Cai et al., 2022). In our study, after 25 years of NT, only minimal changes in SOC were observed, indicating that the NT treatment have reached a steady-state. Considering that the most significant losses of SOC are observed in surface horizons (Wang et al., 2022), the adoption of NT, which encourage C accumulation in the surface layers, has proven to be an effective approach in preserving SOC levels. 4.3. Nitrogen fertilizer effects The application of N fertilization, regardless of the rate, increased SOC levels compared to unfertilized treatments. This effect extended to SOC fractions, including POM-C, Min-C, and POxC. However, consistent with findings from other studies conducted in the Mediterranean region, higher N fertilization rates did not lead to additional increases in SOC levels or any of the studied SOC fractions (L´ opez-Bellido et al., 2010; ´ Alvaro-Fuentes et al., 2013). These results underscore the benefits of moderate N fertilization rates, which enhance system efficiency by reducing the need for excessive mineral fertilizers. Similar to the other treatments examined in this experiment, the increased C inputs resulting from fertilization treatments justify the observed higher levels of POM-C (Salinas-Garcia et al., 1997; Sainju et al., 2003; Russell et al., 2005). These findings are consistent with the results of other fertilization experiments (Lou et al., 2011; Lu et al., 2011; Pareja-S´ anchez et al., 2020) and support the notion proposed by Plaza-Bonilla et al. (2014) that POM-C is a highly responsive fraction to N fertilization. Similar studies have demonstrated that applying appropriate rates of N fertilization enhances soil microbial communities (Thierfelder et al., 2018; Ramírez et al., 2020; Zhang et al., 2021). This microbial stimulation facilitates the decomposition of high-quality plant litter, leading to the production of root exudates and microbial compounds (Knorr et al., 2005; Cotrufo et al., 2013; Rocci et al., 2021). These processes not only directly increase POxC levels (Bolan et al., 2011; Ramírez et al., 2020) but also contribute to the formation of Min-C through mechanisms such as direct sorption and the subsequent physicochemical stabilization of carbon (Panchal et al., 2022; Lei et al., 2023). Nonetheless, under the conditions of this experiment, high rates of N fertilization led to an accelerated decomposition of SOC, resulting in reduced SOC content. This phenomenon is often linked to a decreased C: N ratio, which shifts microbial communities from fungalto bacterialdominated systems, thereby increasing the rate of SOC decomposition (Six et al., 2006; Yuxin et al., 2011; Spohn et al., 2016). These findings highlight the importance of optimizing fertilization practices to strike a balance between maintaining crop productivity and minimizing soil C losses. 5. Conclusion In Mediterranean soils, irrigated maize monocropping under conventional tillage has been shown to decrease SOC levels due to intensified organic matter decomposition and insufficient carbon inputs. Transitioning to legume-maize double-cropping systems has proven to be useful in maintaining SOC levels by increasing C inputs and improving SOC cycling. Similarly, adopting no-tillage practices improves SOC sequestration in surface layers through reduced disturbance and enhanced biological activity linked to carbon stabilization. While nitrogen fertilization supports crop productivity and contributes to carbon inputs, excessive rates accelerate SOC decomposition, highlighting the need for optimized application rates to balance carbon retention and system efficiency. In the face of rising temperatures and climatic challenges in Mediterranean regions, integrating legume-maize double-cropping, notillage, and moderate nitrogen fertilization emerges as a sustainable strategy to maintain SOC levels, enhance microbial function, and J. Fern´ andez-Ortega et al. Soil & Tillage Research 249 (2025) 106496 8 improve soil resilience. CRediT authorship contribution statement Delgado Antonio: Methodology, Investigation. García-L´ opez Ana: Methodology, Investigation. Fern´ andez-Ortega Jesús: Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation. Cantero-Martínez Carlos: Supervision, Investigation, Funding acquisition. ´ Alvaro-Fuentes Jorge: Writing – review & editing, Supervision, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to thank Carlos Cort´ es and Silvia Martí for laboratory and field assistance. This research work was financially supported by the Ministerio de Ciencia e Innovaci´ on of Spain (project AGL2017–84529-C3–3-R;; PhD fellowship PRE2018–084610). Data availability Data will be made available on request. References Adetunji, A.T., Lewu, F.B., Mulidzi, R., Ncube, B., 2017. The biological activities of β-glucosidase, phosphatase and urease as soil quality indicators: a review. J. Soil Sci. Plant Nutr. 17, 794–807. Aguilera, E., Lassaletta, L., Gattinger, A., Gimeno, B.S., 2013. Managing soil carbon for climate change mitigation and adaptation in Mediterranean cropping systems: a meta-analysis. Agric. Ecosyst. Environ. 168, 25–36. Ajwa, H.A., Tabatabai, M.A., 1994. Decomposition of different organic materials in soils. Biol. Fertil. Soils 18, 175–182. Allen, R.G., Pereira, L.S., Raes, D., & Smith, M., 1998. 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