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

Assessing managemen s a egies o ca bon s o age in Medi e anean soils: Double-c opping, no- illage, and ni ogen e iliza ion educ ion Jesús Fe n´ andez-O ega a,* , Jo ge ´ Al a o-Fuen es b , An onio Delgado c , Ana Ma ía Ga cía-L´ opez c , Ca los Can e o-Ma ínez a a Ag icul u al and Fo es Science and Enginee ing Dp . Uni e si y o Lleida - Ag o ecnio CERCA Cen e , A . Alcalde Ro i a Rou e, 191, Lleida 25198, Spain b Soil and Wa e Dp ., Es aci´ on Expe imen al de Aula Dei (EEAD), Spanish Na ional Resea ch Council (CSIC), A d. de Mon a˜ nana, 1005, Za agoza 50059, Spain c Depa men o Ag onomy, Uni e si y o Se ille, C a. U e a km 1, Se ille 41013, Spain ARTICLE INFO Keywo ds: Double-c opping sys em Legume Maize No- illage N e iliza ion Soil o ganic ca bon ABSTRACT In Medi e anean condi ions, he his o ical use o adi ional ag icul u al p ac ices has led o a signi ican loss o soil o ganic ca bon (SOC) and he associa ed bene i s i p o ides. Consequen ly, i becomes impe a i e o explo e e ec i e s a egies ha p omo e he p ese a ion and enhancemen o SOC. Some p omising p ac ices o inc ease SOC a e he use o double-c opping, conse a ion illage, and educed N e iliza ion. The aim o his s udy was o e alua e he combined e ec s o in oducing a legume p io o maize, oge he wi h di e en illage sys ems and mine al N e iliza ion a es on SOC and ela ed ac ions (pa icula e o ganic ma e ca bon, POM-C; mine al- associa ed o ganic ma e ca bon, Min-C; and pe mangana e-oxidizable o ganic ca bon, POxC). Addi ionally, he s udy aimed o in es iga e enzyma ic ac i i ies associa ed wi h he ca bon cycle. The s udy compa ed mono- c opping maize (MC) e sus legume-maize double-c opping (DC) wi h wo illage sys ems (con en ional illage, CT; no- illage, NT), and h ee mine al N e iliza ion a es (ze o, medium and high). The legumes employed we e pea o g ain (2019), e ch o g een manu e (2020), and e ch o o age (2021). The DC inc eased he SOC le el by 10.6 % compa ed o he use o MC, wi h POM-C as he main ac ion in ol ed in his change. Thus, he employmen o DC allowed o he main enance o SOC le els, while he use o MC esul ed in hei educ ion compa ed o he le els obse ed a he beginning o he expe imen . NT exhibi ed highe alues o SOC and i s ac ions POM-C and Min-C. These di e ences we e obse ed only in he 0–10 cm dep h laye s. The use o NT enabled he main enance o SOC compa ed o he ini ial s udied pe iod, while CT educed SOC. The ea men s wi h N e iliza ion achie ed highe alues o SOC and all he s udied ac ions compa ed o he un e ilized ea men . Howe e , a he end o he expe imen , i was ound ha he applica ion o N e iliza ion, especially a high a es, led o a dec ease in SOC. Addi ionally, i was obse ed ha he employmen o DC and NT inc eased he enzyma ic ac i i ies o dehyd ogenase and β-glucosidase. The esul s o his s udy indica e ha he u iliza ion o legume-maize DC, as well as he implemen a ion o NT and educed N e iliza ion, a e use ul s a egies o main ain SOC le els and imp o ing he biological quali y o he soil unde Medi e anean i iga ed condi ions. 1. In oduc ion Soils a e he la ges s o e o e es ial o ganic ca bon (C), wi h an accumula ion g ea e han ha accumula ed by he a mosphe e and plan s combined, and an es ima ed igu e amoun o 1580 G C (Jobb´ agy and Jackson, 2000; Co u o e al., 2019). The e o e, C seques a ion in soils plays a c ucial ole om an en i onmen al pe spec i e, by seques e ing CO 2 om he a mosphe e, and om a soil e ili y pe spec i e by imp o ing he physical, chemical, and biological p op- e ies o he soil (Sainju e al., 2011). The d i e s o soil o ganic ca bon (SOC) changes can be g ouped in o h ee main ca ego ies: 1) clima ic a iables such as p ecipi a ion and empe a u e; 2) soil condi ions such as physical and chemical p ope ies; 3) bio ic p ope ies such as he quan i y and quali y o C inpu s o he soil o soil mic oo ganism Abb e ia ions: CT, con en ional illage; DC, double-c opping; MC, monoc opping; Min-C, mine al-associa ed ca bon; NT, no- illage; NO− 3, ni a e; NH+ 4, ammo- nium; PNP, p-ni ophenol; POM-C, pa icula e o ganic ca bon; POxC, pe mangana e-oxidizable o ganic C; SOC, soil o ganic ca bon; SOM, soil o ganic ma e ; TPF, iphenyl o mazan. * Co esponding au ho . E-mail add ess: [email p o ec ed] (J. Fe n´ andez-O ega). Con en s lis s a ailable a ScienceDi ec Soil & Tillage Resea ch jou nal homepage: www.else ie .com/loca e/s ill h ps://doi.o g/10.1016/j.s ill.2025.106496 Recei ed 18 Janua y 2024; Recei ed in e ised o m 29 Janua y 2025; Accep ed 12 Feb ua y 2025 Soil & Tillage Resea ch 249 (2025) 106496 A ailable online 15 Feb ua y 2025 0167-1987/© 2025 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ). biodi e si y (Luo e al., 2017). These d i e s c ea e an annually balanced sys em as he losses o C o he a mosphe e h ough au o o- phic and he e o ophic espi a ion a e compensa ed by he C gain ob- ained in he soil h ough pho osyn hesis. Howe e , he changes o land use owa ds ag icul u al sys ems and he in ensi ica ion o ag icul u e a e accele a ing he losses o SOC o he a mosphe e (McLauchlan, 2006; Lal, 2007; Ga cía-Palacios e al., 2021). Addi ionally, he cu en a i- abili y in empe a u es and p ecipi a ion u he complica es he si ua- ion. Inc eases in empe a u e can con ibu e o an escala ion in SOC losses h ough accele a ed mic obial decomposi ion and oo espi a ion p ocesses (Rich e e al., 2007; S ewa e al., 2007; Sen hilkuma , 2009). On he o he hand, soil d ying and we ing p ocesses ha e also been shown o inc ease o ganic ma e decomposi ion by p omo ing mic obial ac i i y (Ma eau e al., 2021). This phenomenon has ga ne ed subs an ial a en ion om he scien i ic communi y in ecen decades, e iden in he comp ehensi e models o he clima e cycle p esen ed by he In e go e nmen al Panel on Clima e Change (IPCC, 2014). Wi hin he ag icul u al domain, a p oposed solu ion o main aining SOC in ol es inc easing o ganic ma e o he soil h ough c op esidues. A p omising echnique is he in ensi ica ion o c ops by employing wo cul i a ion cycles pe yea , also known as double- c opping (Zhao e al., 2018). The e ec s o hese in ensi ied sys ems on SOC a y depending on he c op species and esidue managemen p ac ices. Double-c opping wi h legume-ce eal combina ions and e aining esidues on he ield has been shown o po en ially inc ease SOC (S agna i e al., 2017; Jian e al., 2020). Howe e , i c op esidues a e emo ed o insu icien e iliza ion is applied, i may lead o a educ ion in SOC (Heggens alle e al., 2008; Ma esma e al., 2019). None heless, he e is limi ed esea ch a ailable indica ing he ad an age o double-c opping in inc easing SOC con en . Conse a ion illage and op imized ni ogen (N) e iliza ion a e also wo in e es ing p ac ices ha ha e been ex ensi ely s udied, showing a signi ican impac on SOC dynamics. Se e al s udies pe o med in he Medi e anean egion ha e demons a ed ha he implemen a ion o conse a ion illage may esul in a di ec inc ease in SOC, wi h an a e age o 0.40–0.50 Mg C ha −1 yea −1 obse ed in he op 30 cm o he soil p o ile (Plaza-Bonilla e al., 2010; ´ Al a o-Fuen es e al., 2012; Mazzoncini e al., 2016). Howe e , he SOC seques a ion p ocess has limi s, and hey a e closely ela ed o soil p ope ies, clima e condi ions, and c op complexi y. A e a change in he illage sys em, i akes abou 15–50 yea s o each an equilib ium le el (s eady-s a e) (Folle , 2001; Wes and Pos , 2002; Smi h and Chalk, 2020). This equilib ium le el also depends on he wa e egime o he expe imen . ´ Al a o-Fuen es and Paus ian (2011) obse ed ha o he same clima e condi ions, he Cen u y model p edic ed a SOC seques a ion du a ion o 90 yea s in i iga ed sys ems, and 70 yea s in ain ed sys ems. Such dispa i ies un- de sco e he impo ance o in es iga ing each speci ic c opping sys em wi hin i s espec i e c opping a ea. N e iliza ion also has a di ec e ec on he soil C balance due o i s e ec on inc easing biomass p oduc ion (Sainju e al., 2002; Bolinde e al., 2020). Howe e , excessi e N e iliza ion may also lead o inc eased o ganic ma e mine aliza ion and he elease o C in o he a mosphe e, as well as po en ial N loss h ough leaching (Mazzoncini e al., 2011). The biological quali y o soils is in ica ely ied o hei capaci y o o ganic ma e mine aliza ion and, consequen ly, SOC seques a ion. Enzyma ic ac i i ies, commonly u ilized in s udies, se e as aluable indica o s o soil biological quali y, displaying co ela ions wi h di e en SOC ac ions and a es o o ganic ma e mine aliza ion. (Moeskops e al., 2010; Zhang e al., 2021). Dehyd ogenase ac i i y, being exclusi ely measu ed wi hin he cells o li ing o ganisms, se es as a eal- ime indica o o soil biological ac i i y and espi a ion (Li e al., 2019). Con e sely, β-glucosidase ac i i y is an ex acellula enzyme wi h a cen al ole in cellulose deg ada ion. Since i s subs a e is a ely limi ed, luc ua ions in his enzyma ic ac i i y a e asc ibed o al e a ions in soil physical and chemical p ope ies (Ei azi and Taba- abai, 1988; Tan e al., 2021). Thus, s udying enzyme ac i i ies in conjunc ion wi h SOC can gi e a deepe unde s anding o he impac s o ag icul u al managemen on SOC changes. I is impe a i e o in es iga e cul i a ion echniques ha no only main ain SOC bu also sus ain c op yields, as highligh ed by se e al au ho s (Blanco-Canqui, 2022; an de Pol e al., 2022). Due o he mul i ude o ac o s in ol ed in he cu en p ocess o SOC loss, con- duc ing s udies ac oss di e se geog aphical a eas becomes necessa y (Na a o-Ped e˜ no e al., 2021). These s udies should encompass comp ehensi e c op managemen , including di e en cul i a ion sys- ems, soil p ac ices, and e ilize a es. The e o e, he objec i es o his s udy we e: i) o assess he impac o legume-maize double-c opping, in conjunc ion wi h di e en illage sys ems and mine al N e iliza ion a es, on SOC con en ; ii) o analyze how he main SOC ac ions a e a ec ed by hese changes in c opping managemen ; and iii) o de e mine he ac i i ies o dehyd ogenase and β-glucosidase enzymes and hei ela ionships wi h he obse ed SOC changes. The ini ial hypo hesis sugges s ha he use o double-c opping, conse a ion illage, and educed ni ogen a es would esul in he highes ca bon accumula ion, leading o he mos signi ican inc eases in SOC le els. Mo eo e , he soil condi ions c ea ed by hese combined managemen s a egies would os e an en i onmen conduci e o highe enzyme ac i i ies, he eby e lec ing imp o ed soil quali y. 2. Ma e ial and me hods 2.1. Expe imen al design and managemen p ac ices The s udy was conduc ed in no heas e Spain, in he Ag amun municipali y (41◦48 ′ N, 1◦07 ′ E, 330 m asl). The egion is ypical o a semia id Medi e anean d yland wi h a con inen al end. In he las 30 yea s, he a e age annual p ecipi a ion has been 442 mm, he a e age annual empe a u e has been 14.6 ◦C, and he annual po en ial e apo- anspi a ion (PET) is 855 mm. The a e age annual empe a u e du ing he h ee-yea du a ion o he expe imen was 15.9 ⁰C, whe eas i had been 14.3 ⁰C du ing he 3 yea s p io o he expe imen (Fig. 1a). The a e age soil mois u e (0–10 cm dep h) o bo h he expe imen al pe iod and he pe iod be o e he expe imen was 25.3 and 18.3 % espec i ely (Fig. 1b). Acco ding o he Soil Su ey S a (2014), he soil is classi ied as Typic Xe o lu en s. The main physiochemical p ope ies a e p esen ed in Table 2. The expe imen al ield was es ablished in 1996 o compa e h ee a es o mine al N (0, 60 and 120 kg N ha −1 ) and h ee illage sys ems (con en ional illage, CT; minimum illage, MT; no- illage, NT) unde ain ed ba ley monoc opping condi ions (Ang´ as e al., 2006) (Table 1). In 2015, he expe imen was ans o med o i iga ed condi ion using Fig. 1. Daily ai empe a u e (a) and soil olume ic mois u e (b) o pe iod 0 (2015–2017) and pe iod 1 (2019–2021). J. Fe n´ andez-O ega e al. Soil & Tillage Resea ch 249 (2025) 106496 2 solid se sp inkle s spaced a 18 ×18 m, and he c op was changed o maize (Zea mays L.) monoc opping (Pa eja-S´ anchez e al., 2017). The illage ea men s we e main ained, and mine al N e iliza ion a es we e adap ed o maize c op (0, 200, 400 kg N ha −1 ) wi h he same expe imen al layou as he p e ious ain ed expe imen . In 2018, o enhance he s udy o c op di e si ica ion and i s in e ac ion wi h illage and N e iliza ion, he plo s we e di ided in o a spli -plo design measu ing 3 m wide and 48 m long. This allowed o a compa ison be- ween wo c opping sys ems (Cs): monoc opping maize (MC) and double-c opping legume-maize (DC). Soil illage ea men s (Till) we e he same as hose used p e iously, al hough only he wo mos di e en ea men s (CT and NT) we e used o his s udy. The N e iliza ion a es (Fe ) we e ze o (0 kg N ha −1 ), medium (200 kg N ha −1 ), and high (400 kg N ha −1 ) o MC while assuming he biological ixa ion o he legume, he a es we e adjus ed o ze o (0 kg N ha −1 ), medium (150 kg N ha −1 ), and high (300 kg N ha −1 ) o DC. Consequen ly, he c op di e si ica ion ea men spanned 3 yea s, whe eas he illage and N e iliza ion ea men s ex ended o e 25 yea s by he conclusion o he cu en expe imen . Fo MC maize, a long-cycle maize cul i a (FAO 700, Pionee ’s P1570 hyb id) was used. Fo DC a sho -cycle maize cul i a (FAO 400, Pionee ’s P0312 hyb id) was plan ed as summe c op and a legume as win e c op. The legumes we e: pea used o g ain (Pisum sa i um L., a . Fu ious) du ing he 2018–2019 season; and e ch (Vicia sa i a L., a . P on i esa) employed as g een manu e du ing 2019–2020 and o age du ing 2020–2021. Th oughou he h ee-yea s udy pe iod, MC maize was sown in Ap il; DC maize in ea ly June; and he legume om Decembe o Janua y. The sowing a e in maize (MC and DC) was 90,000 seeds ha −1 wi h a sepa a ion be ween lines o 73 cm. In he case o legumes, a densi y o 100 plan s m −2 was used o pea and 267 plan s m −2 o e ch. The CT ea men consis ed o subsoile (35 cm dep h) ollowed by one pass o o o ille (15 cm dep h) and one pass o olle be o e plan ing, wi h almos 100 % o he c op esidues inco po a ed in o he soil. NT plo s we e sp ayed wi h he bicide, 1.5 L ha −1 o 36 % glypho- sa e [N-(phosphonome hyl)-glycine] wi hou dis u bing he soil. Plan ing was done wi h a pneuma ic ow di ec seeding machine equipped wi h double disc u ow opene s (model P osem K, Sol`a, Cala , Spain). N e ilize was manually applied, and exclusi ely o he maize c op. In MC, p e-sowing u ea (46 % N) e iliza ion ook place in Ap il-May. The u ea was b oadcas ed on he soil su ace and inco po a ed h ough illage in CT, while i was le on he g ound in NT. The p e- sowing e iliza ion a e was 50 and 100 kg N ha −1 o he medium and high a es, espec i ely. Fu he mo e, bo h in MC and DC, wo op- d essing e iliza ions we e pe o med in s ages V3-V5 (May in MC, and la e June in DC) and V7-V8 (June in MC, and July in DC). These op- d essing applica ions in ol ed ammonium ni a e (34.5 % N) a 75 and 150 kg N ha −1 o he medium and high a es, espec i ely. A he onse o each g owing season, mine al P and K e ilize s we e applied o ul ill he nu i ional demands o maize and legumes, in acco dance wi h he s anda d yields obse ed in he egion. I iga ion was conduc ed om Ma ch o Oc obe based on he c op’s wa e equi emen s. The i iga ion schedule was de e mined by he c op wi h he highes wa e demand, which was es ima ed weekly by sub ac ing he e ec i e p e- cipi a ion (75 % o he o al weekly p ecipi a ion) om he c op e apo anspi a ion (ETc) (Das ane, 1978). The ETc was de e mined using he FAO Penman-Mon ei h me hodology wi h me eo ological da a ob ained om a nea by wea he s a ion in he icini y o he ield expe imen . This da a was hen mul iplied by he c op coe icien (Kc) speci ic o he c op, de i ed as a unc ion o he mal ime (Allen e al., 1998). Table 1 Managemen his o y o he expe imen al ield. Sys em: Rain ed S a ing yea 1996   C op: Ba ley T ea men s Ni ogen (Kg N ha −1 ) Ze o - 0  Medium - 60  High - 120  Tillage Con en ional illage  No illage  Sys em: Sp inkle i iga ion    S a ing yea 2015     C op: Maize T ea men s Ni ogen (Kg N ha −1 ) Ze o - 0   Medium - 200   High - 400   Tillage Con en ional illage   No illage   2019 T ea men s C op: Maize Legume-Maze Ni ogen (Kg N ha −1 ) Ze o 0 0 Medium 200 150 High 400 300 Tillage Con en ional illage   No illage   C opping di e si ica ion Monnoc opping maize   Legume-Maize Double-c opping   Table 2 Soil p ope ies o he Ap ho izon (0–28 cm dep h) in 1996. Ini ial soil o ganic ca bon con en (SOC i ) (1996) and soil o ganic ca bon con en (SOC) (0–30 cm) in h ee illage sys ems (con en ional illage, CT; no- illage, NT) in 2017. Soil p ope ies Soil classi ica ion*Typic Xe o lu en 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 Wa e e en ion (g g −1 ) −33 kPa 0.16 −1500 kPa 0.05 Soil Tex u e (g kg −1 ) Sand (2–0.05 mm) 308 Sil (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 * Acco ding o he USDA classi ica ion (Soil Su ey S a , 2014) J. Fe n´ andez-O ega e al. Soil & Tillage Resea ch 249 (2025) 106496 3 2.2. Soil sampling and analysis The ini ial soil sampling was conduc ed in No embe 2018 p io o he s a o he expe imen . The inal soil sampling was conduc ed in No embe 2021, ollowing he ha es o maize. Soil samples we e aken in wo obse a ions pe plo a : 0–5, 5–10, 10–20, 20–30, 30–40 cm o dep h. To al soil o ganic ca bon (SOC), pe mangana e-oxidizable o ganic C (POxC), pa icula e o ganic ma e ca bon (POM-C) and mine al-associa ed o ganic ca bon (Min-C) we e analysed. These spe- ci ic ac ions we e chosen due o hei a ying sensi i i y o changes esul ing om di e en ag icul u al managemen p ac ices (´ Al a o-Fuen es e al., 2014). SOC de e mina ion was pe o med ollowing he me hodology o Walkley and Black (1934), using a 1 g soil subsample, p e iously d ied and sie ed a 2 mm. POM-C and Min-C ac ions we e analysed acco ding o Camba della and Ellio (1992) wi h a modi ica ion de eloped o ou condi ions by Pa eja S´ anchez e al. (2020). B ie ly, wen y-g am subsamples o soil om each dep h and plo we e dispe sed in 100 mL o 5 g L −1 sodium hexame aphos- pha e o 15 h on a ecip ocal shake . Then he samples we e passed h ough a 50 μ m sie e o sepa a e he POM and Min-C. The ma e ial passing h ough he sie e (Min-C) was collec ed in aluminium pans and o en d ied a 50 ◦C. The we oxida ion me hod o Walkley and Black (1934) was hen used o measu e he C concen a ion in he Min-C ac ion. The POM-C con en was de e mined as he di e ence be- ween o al SOC con en and Min-C con en . POxC was de e mined ac- co ding o Weil e al. (2003). SOC con en s we e de e mined based on mass pe uni a ea, calcula ed by mul iplying he ca bon concen a ion alues ob ained h ough he oxida ion me hod by he co esponding soil bulk densi y alues. Addi ionally, SOC s ock (kg C ha −1 ) was adjus ed o equi alen soil mass acco ding o he p ocedu e ou lined by Elle and Be any (1995) o he 0–40 cm soil dep h in e al. The annual SOC seques a ion a e (ΔSOC a e) (kg C ha −1 y −1 ) (0–40 cm soil dep h) was compu ed o each ea men om 2018 o 2021. The objec i e conce ning his pa ame e was o assess he con inued use o i iga ed maize e sus he p ac ice o legumes-maize double-c opping. Soil sampling o enzyma ic ac i i ies was pe o med in wo obse - a ions pe plo a 0–5, 5–10, 10–20, 20–30 cm soil dep hs, exclusi ely in he CT and NT plo s. Soil o wo obse a ions we e mixed and passed h ough a 2 mm sie e o ob ain a ep esen a i e and unique sample a di e en dep hs. Dehyd ogenase ac i i y was de e mined om 3 g o esh soil ollowing he me hod o Casida e al. (1964) based on he de e mina ion o iphenyl o mazan (TPF) p oduced om 2,3,5- i- phenyl e azolium chlo ide a e 24 h incuba ion a 37 ◦C in he da k. β-glucosidase ac i i y was analysed om 1 g o esh soil ollowing he me hod o Ei azi and Taba abai (1988) based on he de e mina ion o p-ni ophenol (pNP) a e incuba ion wi h p-ni ophenyl-β -D-glucoside o 1 h. 2.3. Ca bon inpu s C op esidue samples we e aken jus a e ha es ing. The me hod o sampling a ied depending on he ype o c op. Fo bo h MC and DC maize, plan samples om 2-m-long cen al ows we e aken in h ee obse a ions pe plo . Fo pea, 0.5-m-long o wo adjacen ows we e sampled a wo obse a ions pe plo . Fo e ch 0.36 m 2 o plan s we e cu a he soil su ace le el in wo a eas o each plo . Only he abo e-g ound biomass o he c op was conside ed o he C inpu s, as he g ain was ha es ed o sale and he oo s we e no sampled. Simila ly, he e ch c op in 2021 was no conside ed in he calcula ions as i was expo ed as o age. The biomass o all c ops was d ied in an o en a 60 ⁰C o 48 h, h eshed and weighed excluding he g ain, he ea e e e ed as c op esidues. The C con en was de e mined by d y combus ion (model T uspec CN, LECO, S Joseph, MI, USA). C inpu s we e calcula ed by mul iplying he biomass o he ag icul u al esidues by hei C con en . 2.4. S a is ical analysis Da a we e checked o no mali y, homoscedas ici y and se ial inde- pendence by Shapi o-Wilk, Ba le , and Du bin-Wa son es espec- i ely. Ou lie s we e checked using he G ubbs es wi h a s a is ical con idence le el o 95 %. Any da a no passing ei he es equi ed addi ional ans o ma ion. Fo C inpu s and enzyma ic ac i i ies da a analysis, a epea ed measu es analysis o a iance (ANOVA) was ca ied ou wi h he c opping sys em, illage and N e iliza ion, yea , and hei in e ac ions as e ec s. Fo SOC and i s ac ions, and o SOC seques- a ion a e, ANOVA was conduc ed wi h he ea men s o c opping sys em, illage and N e iliza ion, dep h, and hei in e ac ions as e - ec s. S a is ical analyses we e pe o med wi h he s a is ical package JMP p o 16 (SAS Ins i u e Inc., 2021) and S a g aphics Cen u ion 18 (S a g aphics Technologies Inc., 2018). 3. Resul s 3.1. Ca bon inpu s and SOC The esul s o his expe imen co espond o he pe iod 2019–2021 (P1). To explain he obse ed changes in SOC du ing his ime, da a on C inpu s om he p e ious s udy pe iod (P0, 2015–2017) by Pa eja S´ anchez e al. (2020) a e also p esen ed (Fig. 2, P0). Du ing P1, he h ee ea men s s udied had a signi ican impac on C inpu s (Table 3). Among he c opping sys ems, DC p oduced he highes C inpu alues, pa icula ly when combined wi h NT and medium o high N e iliza ion a es. The a e age C inpu s o hese combina ions anged be ween 7086 and 8257 kg C ha⁻¹ (Fig. 2). In con as , he lowes C inpu s we e consis en ly obse ed in he MC-CT combina ion, ega dless o he N e iliza ion a e, wi h an a e age o 3323 kg C ha⁻¹ ac oss all a es. When compa ing C inpu s ac oss pe iods, he inpu s om P0 we e simila o hose o he MC ea men du ing P1, wi h a e ages o 3100 kg C ha⁻¹ (P0) and 3650 kg C ha⁻¹ (P1), espec i ely. By he conclusion o he expe imen in No embe 2021, SOC le els we e signi ican ly in luenced by all h ee ea men s (Table 3). The DC sys em showed he highes SOC concen a ion (9.4 g C kg⁻¹ soil). Rega ding illage managemen , NT esul ed in he highes SOC le els (9.8 g C kg⁻¹ soil). Fo N e iliza ion, signi ican di e ences we e obse ed be ween he e ilized ea men s and he un e ilized con ol, wi h a mean SOC concen a ion o 8.7 g C kg⁻¹ soil ac oss e ilized ea men s. Howe e , no signi ican di e ences we e ound be ween medium and high N e iliza ion a es. Fig. 2. Tillage (CT, con en ional illage; NT, no- illage) and a e o N e il- iza ion (Ze o, Med and High: 0, 200, and 400 kg N ha −1 o MC; 0, 150, and 300 kg N ha −1 o DC), e ec s on C inpu s (pe iod 0); and c opping sys em (MC, monoc opping sys em; DC double-c opping sys em), illage and a e o N e iliza ion e ec s on C inpu s (pe iod 1). Fo pe iod 0, he e a e no signi ican di e ences among ea men s; o pe iod 1, he a ious lowe case le e s indi- ca e signi ican di e ences among ea men s a p<0.05. The e ical ba s indica e he s anda d e o . J. Fe n´ andez-O ega e al. Soil & Tillage Resea ch 249 (2025) 106496 4 When compa ing SOC le els a he beginning and he end o he expe imen , he ΔSOC a e was also signi ican ly in luenced by all h ee ea men s s udied (Table 4). Unde he DC sys em, he C s ock emained s able compa ed o 2018, while he MC sys em expe ienced a decline in C s ock a a a e o 178 kg C ha⁻¹ yea ⁻¹ . In e ms o illage managemen , NT e ec i ely p ese ed soil C s ock, whe eas CT led o a educ ion o 199 kg C ha⁻¹ yea ⁻¹ . Rega ding ni ogen e iliza ion, he applica ion o N, especially a high a es, caused a educ ion in C s ock, wi h de- c eases o 90 and 157 kg C ha⁻¹ yea ⁻¹ o medium and high N a es, espec i ely. In con as , he un e ilized ea men main ained C s ock le els. SOC ac ions e ealed signi ican changes associa ed wi h he c opping sys em. Speci ically, he use o DC esul ed in a 19.5 % inc ease in POM-C and a 12.7 % inc ease in POxC compa ed o MC. The illage ea men also impac ed he POM-C and Min-C ac ions, wi h NT showing POM-C alues 44.4 % highe han CT. N e iliza ion in luenced all s udied SOC ac ions. As wi h o al SOC con en , signi ican di e ences we e obse ed only be ween e ilized ea men s and he un e ilized con ol, wi h no no able di e ences be ween medium and high e iliza ion a es. On a e age, e ilized ea men s showed inc eases o 13.8 % in POM-C, 11.0 % in Min-C, and 12.5 % in POxC compa ed o he un e ilized ea men (Table 4). A signi ican in e ac ion be ween he illage sys em and sampling dep h was also obse ed o bo h o al SOC and i s ac ions (Table 4). In he opsoil laye (0–5 cm), NT had he highes SOC concen a ion (18.5 g C kg⁻¹ in NT s. 11.0 g C kg⁻¹ in CT) (Fig. 3a). A 5–10 cm dep h, NT also main ained signi ican ly highe alues han CT. Howe e , a deepe laye s (10–40 cm), no signi ican di e ences we e ound be- ween illage ea men s, wi h an a e age SOC concen a ion o 4.5 g C kg⁻¹ . Fo POM-C, he highes alues we e obse ed in he su ace laye (0–10 cm) unde NT, wi h concen a ions o 11.5 and 6.4 g C kg⁻¹ a 0–5 cm and 5–10 cm dep hs, espec i ely. No signi ican di e ences we e ound be ween illage ea men s a in e media e o deepe laye s (Fig. 3b). Min-C alues we e also highe unde NT in he 0–10 cm ange, while simila alues we e eco ded a g ea e dep hs ac oss all illage ea - men s (Fig. 3c). Fo POxC, signi ican di e ences be ween ea men s we e obse ed only in he mos supe icial laye (0–5 cm), whe e NT eco ded a mean alue o 760.5 mg C kg⁻¹ , signi ican ly highe han CT a 572 mg C kg⁻¹ (Fig. 3d). 3.2. Soil enzyma ic ac i i ies The e ec o c opping on dehyd ogenase ac i i y was ound o depend on illage ea men s ac i i y as e ealed by he signi ican in e ac ion be ween ac o s. Dehyd ogenase ac i i y was no ably highe in he NT sys em, especially when combined wi h DC, eaching 5.5 μ g TPF g −1 d y soil h −1 (Fig. 4a). The signi ican di e ences ound in β-glucosidase ac i i y we e a ibu ed o indi idual ac o s o c opping and illage sys ems (Table 3). β-Glucosidase ac i i y in he DC sys em was 23 % highe compa ed o MC (Fig. 4b). Addi ionally, NT esul ed in a 53 % inc ease in β-glucosidase ac i i y (Fig. 4c). 4. Discussion 4.1. Legume-maize double-c opping e ec s The indings o his s udy demons a e ha eplacing win e allow in monoc opping maize sys ems wi h a legume signi ican ly inc eases SOC le els. I is well-es ablished ha highe C inpu s consis en ly ac as he p ima y d i e o SOC accumula ion ac oss di e se en i onmen s (Kong e al., 2005; Liu e al., 2014). In his expe imen , he highes C inpu s we e obse ed in he DC sys em, p ima ily due o he p esence o wo c op phases pe yea and he imp o emen s in DC maize yields p o ided by he p eceding legume phase (Talukde e al., 2022; Fe n´ andez-O ega e al., 2023b). This inding was u he suppo ed by a s a is ically sig- ni ican co ela ion be ween SOC le els and soil inpu s, modeled h ough a wo-deg ee polynomial eg ession (p <0.001, ² =0.22). The analysis o C ac ions e ealed ha he p ima y ac ion a ec ed by he use o DC was POM-C. This ac ion is composed o Table 3 Analysis o a iance (p- alues) o ca bon inpu s (C-inpu ), soil dehyd ogenase ac i i y (DHA) and soil β-Glucosidase ac i i y (β-Glu) as a ec ed by c opping sys em, illage, a e o N e iliza ion, yea and hei in e ac ions. Sou ce o a ia ion C inpu s DHA β-Glu C opping sys em (Cs) <0.001 NS <0.01 Tillage (Till) <0.001 NS <0.001 N e iliza ion (Fe ) <0.001 NS NS Cs ⋅ Till NS NS NS Cs ⋅ Fe NS <0.05 NS Till ⋅ Fe NS <0.001 <0.001 Cs ⋅ Till ⋅ Fe <0.05 NS NS Yea <0.001 NS NS Yea ⋅ Cs <0.001 NS NS Yea ⋅ Till NS <0.001 NS Yea ⋅ Fe <0.001 NS NS Yea ⋅ Cs ⋅ Till NS NS NS Yea ⋅ Cs ⋅ Fe <0.05 NS NS Yea ⋅ Till ⋅ Fe NS NS NS Yea ⋅ Cs ⋅ Till ⋅ Fe NS NS NS NS, no signi ican Table 4 Analysis o a iance a he end o he expe imen (No embe 2021) o soil o ganic ca bon (SOC), pa icula e o ganic ma e ca bon (POM-C), mine al- associa ed ca bon (Min-C), pe mangana e-oxidizable o ganic C (POxC) con- cen a ion and soil o ganic ca bon seques a ion a e (ΔSOC a e) as a ec ed by c opping sys em (MC, monoc opping sys em; DC, double-c opping sys em), illage (CT, con en ional illage; NT, no- illage), a e o N e iliza ion (Ze o, Med and High: 0, 200, and 400 kg N ha −1 o MC; 0, 150, and 300 kg N ha −1 o DC), dep h, and hei in e ac ions. Nega i e alues o ΔSOC a e imply a dec ease in soil ca bon s ock compa ed o he yea 2018. Sou ce o a ia ion SOC POM-C Min-C POxC ΔSOC a e  g C kg soil −1 mg C kg soil −1 kg C ha −1 y −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 Ze o 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      C opping sys em (Cs) <0.01 <0.001 NS <0.05 <0.001 Tillage (Till) <0.001 <0.001 <0.001 NS <0.001 N e iliza ion (Fe ) <0.05 NS <0.05 NS <0.05 Cs ⋅ Till NS NS NS NS NS Cs ⋅ Fe NS NS NS NS NS Till ⋅ Fe NS NS NS NS NS Cs ⋅ Till ⋅ Fe NS NS NS NS NS Dep h <0.001 <0.001 <0.001 <0.001 <0.001 Dep h ⋅ Cs NS NS NS NS NS Dep h ⋅ Till <0.001 <0.001 <0.001 <0.05 NS Dep h ⋅ Fe NS NS NS NS NS Dep h ⋅ Cs ⋅ Till NS NS NS NS NS Dep h ⋅ Cs ⋅ Fe NS NS NS NS NS Dep h ⋅ Till ⋅ Fe NS NS NS NS NS Dep h⋅ Cs ⋅ Till ⋅ Fe NS NS NS NS NS NS, no signi ican . Di e en lowe case le e s indica e signi ican di e ences among ea men s a p<0.05. J. Fe n´ andez-O ega e al. Soil & Tillage Resea ch 249 (2025) 106496 5 undecomposed o pa ially decomposed ma e ials, including mic obial biomass, plan esidues, and oo emnan s (Six e al., 2001; Von Lü zow e al., 2008). I exhibi s he mos apid changes in esponse o a ia ions in C inpu s, ypically wi hin weeks o yea s (Culman e al., 2012; Plaza-Bonilla e al., 2014; Wooli e and Jagadamma, 2023). Mo eo e , i has been iden i ied as a eliable p edic o o changes in he con en and quali y o soil o ganic ma e , as well as a signi ican sou ce o po en ially mine alizable o ganic ma e in he soil (Semeno e al., 2019). In his s udy, POM-C le els we e signi ican ly highe in he DC sys em, u he ein o cing he idea ha changes in SOC a e p ima ily d i en by di e ences in C inpu s. In con as , he use o DC did no lead o signi ican di e ences compa ed o MC in he Min-C ac ion. Min-C consis s o single molecules o mic oscopic agmen s o o ganic ma e- ial leached om plan ma e ial o ans o med by soil bio a (La allee e al., 2020; Rocci e al., 2020). Due o he long mean esidence ime o c op esidues, no iceable inc eases in his ac ion ypically ake decades o cen u ies (Camba della and Ellio , 1992; S i as a a e al., 2016). Min-C is also p o ec ed om decomposi ion by associa ions wi h soil mine als, such as chemical bonds wi h mine al su aces and occlusion wi hin mic opo es o small agg ega es (<50–63 µm), limi ing i s accessibili y o decompose s and enzymes. As a esul , c op managemen changes ha e a slow impac on Min-C le els (To sche e al., 2018; Co u o e al., 2019). In his s udy, he h ee-yea DC implemen a ion was insu icien o p oduce signi ican di e ences in Min-C compa ed o MC sys ems. The use o DC inc eased he con en o POxC compa ed o MC. POxC ep esen s he ea ly decomposi ion p oduc s o plan and aunal biomass, oo exuda es, and mic obial biomass (Bolan e al., 2011; Mo ow e al., 2016). I s apid o ma ion makes POxC he mos ac i e SOC componen (Zhang e al., 2021), o en used as an e ec i e indica o o sho - e m changes in SOC ac ions (Plaza-Bonilla e al., 2014; Gaba ´ on-Galeo e e al., 2015). The inco po a ion o easily deg adable legumes in luences he deg ada ion o o ganic ma e , con e ing s able C pools in o mo e labile C o ms (Li e al., 2019). Fu he mo e, he DC sys em exhibi ed highe dehyd ogenase and β-glucosidase ac i i ies. Dehyd ogenase ac i i y, which exis s only in in ac cells, e lec s he le el o ac i e mic obial ac i i y a a gi en ime (Veum e al., 2014). I is associa ed wi h mic oo ganisms esponsible o b eaking down o ganic ma e ials (Ross, 1971; Bol on e al., 1985; Pio- owska-Długosz e al., 2022), leading o apid inc eases in soil POxC (Leno e al., 2021) and explaining he highe POxC alues obse ed unde DC. Simila ly, β-glucosidase ac i i y inc eases wi h he addi ion o easily deg adable legume esidues (Ei azi and Taba abai, 1990; Na as e al., 2011). This enzyme plays a key ole in he inal b eakdown o plan deb is ich in cellulose (Tu ne e al., 2002). Highe β-glucosidase ac i i y has been linked o inc eased le els o Min-C and POxC in a ious s udies (Ajwa and Taba abai, 1994; Whi b ead e al., 2000; Haynes, 2005). Combined, hese enzyma ic ac i i ies sugges ha legumes in he Fig. 3. Soil o ganic ca bon (SOC) (a), pa icula e o ganic ma e ca bon (POM-C) (b), mine al-associa ed ca bon (Min-C) (c) and pe mangana e-oxidizable o ganic C (POxC) (d) as a ec ed by illage (CT, con en ional illage; NT, no- illage) a di e en soil dep hs a he end o he s udy (2021). Wi hin a soil laye Di e en lowe case le e s indica e signi ican di e ences among ea men s a p<0.05. J. Fe n´ andez-O ega e al. Soil & Tillage Resea ch 249 (2025) 106496 6 DC sys em enhance mic obial ac i i y and, consequen ly, imp o e C dynamics and ixa ion in he soil (Visse and Pa kinson, 1992; de la Paz Jimenez e al., 2002). The con inuous use o MC unde i iga ed condi ions led o a loss o SOC compa ed o he s a o he expe imen in 2019. Al hough he C inpu s om MC du ing his pe iod (P1, 2019–2021) we e compa able o hose epo ed in he p e ious s udy by Pa eja-S´ anchez e al. (2020) (P0, 2015–2017), conduc ed in he same expe imen al ield unde iden ical illage and N ea men s, hey we e insu icien o main ain SOC le els. I has been demons a ed ha sus ained i iga ion can inc ease enzyma ic ac i i y and accele a e he mine aliza ion o o ganic ma e , leading o SOC educ ions, pa icula ly unde equen cycles o soil we ing and d ying (Nunes e al., 2007; Ma eau e al., 2021). Fu he - mo e, nume ous global s udies ha e consis en ly iden i ied a s ong co ela ion be ween declining SOC and inc eases in empe a u e and humidi y (Sen hilkuma , 2009; Mo ug´ an-Co onado e al., 2020; Ma iappan e al., 2022). This decline is p ima ily d i en by accele a ed decomposi ion a es (Da idson e al., 2000; Conan e al., 2008) and inc eased esidue mine aliza ion (Rus ad e al., 2001; Ga cía-Palacios e al., 2021). In his pa icula s udy, he annual mean empe a u e and maximum empe a u e we e 1.6 ◦C and 3.5 ◦C highe , espec i ely, compa ed o he p e ious pe iod. Addi ionally, soil mois u e was, on a e age, 7 % highe . Thus, he con inued use o in ensi e i iga ion sys ems, combined wi h inc eased empe a u e and soil mois u e, has been iden i ied as he p ima y ac o s con ibu ing o he obse ed educ ion in SOC wi hin he MC sys em. In con as , using legumes be o e maize cul i a ion helped main ain SOC le els. Resea ch has shown ha inco po a ing legumes in o o a- ions can help p ese e SOC, especially when esidues added o he soil ha e a low C/N a io (Paus ian e al., 2019; an de Pol e al., 2022). DC no only inc eases C inpu s bu also enhances soil agg ega e o ma ion (Talukde e al., 2023). This imp o ed s uc u e physically p o ec s C inpu s om mic obial decomposi ion (Negassa e al., 2015; Vi k e al., 2022). Addi ionally, legumes al e he soil mic obiome’s composi ion and s a egies, enabling e icien decomposi ion and u iliza ion o plan -de i ed C sou ces a he han elying solely on exis ing o ganic C (Malik e al., 2020). These e ec s p omo e e icien ans o ma ions among SOC ac ions, leading o s able o ganic C seques a ion h ough mic obial p ocesses (Li e al., 2023). Howe e , despi e he highe decomposi ion a es obse ed in his s udy, he inc eased C inpu s unde DC we e insu icien o augmen C s ocks compa ed o he expe imen ’s beginning, sugges ing ha he DC sys em has eached a s eady-s a e (Powlson e al., 2011; Nicoloso e al., 2016; an de Pol e al., 2022). 4.2. Tillage sys ems e ec s In line wi h he ini ially p oposed hypo hesis, he NT sys em showed an inc ease in SOC and i s ac ions, POM-C and Min-C, compa ed o CT. Simila o he c opping sys em ea men , he highe le els o POM-C in he NT sys em a e p ima ily a ibu ed o he inc eased C inpu s obse ed in NT, ela i e o CT. Nume ous s udies unde compa able condi ions ha e shown ha NT sys ems imp o e soil physical p ope ies, including he o ma ion o soil mac oagg ega es, enhanced soil s uc- u e, and inc eased soil wa e con en (Lampu lan´ es and Can e - o-Ma ínez, 2003; Pa eja-S´ anchez e al., 2017; Talukde e al., 2022), which in u n suppo s highe biomass yields. Fu he mo e, he inc ease in POM-C is pa icula ly associa ed wi h he NT sys em, mainly due o he be e p o ec ion p o ided by soil agg ega es in his illage sys em and he educed bu ial o c op esidues compa ed o CT (Zube and Villamil, 2016; Kan e al., 2020; Vilakazi e al., 2022). These di e ences a e especially no iceable in he uppe soil laye s, whe e he highes concen a ion o esidues accumula es and whe e his ac ion is mo e sensi i e o illage ope a ions (Wande and Bida , 2000; Li e al., 2022). In he case o Min-C, he e ha e been limi ed s udies on illage ha Fig. 4. Dehyd ogenase ac i i y as e ec s o he in e ac ion o c opping sys em (MC, monoc opping sys em; DC, double-c opping sys em) and illage (CT, con en- ional illage; NT, no- illage) (a), and β-glucosidase ac i i y as a ec ed by he c opping sys em (b) and illage (c). The a ious lowe case le e s indica e signi ican di e ences among ea men s a p <0.05. The e ical ba s indica e he s anda d de ia ion. J. Fe n´ andez-O ega e al. Soil & Tillage Resea ch 249 (2025) 106496 7 epo ed signi ican esul s (Poeplau and Don, 2015; Wooli e and Jagadamma, 2023). This is due o he ecalci an na u e o his ac ion o C, as well as he ypically la ge pool size in mine al soils, which leads o e y slow changes (Rocci e al., 2021). In ou s udy, al hough he p esen expe imen las ed 3 yea s, he 25 yea s o main aining he same illage p ac ices in he o al du a ion o he long- e m expe imen ha e allowed us o obse e he highes le els o Min-C in he NT. The main jus i ica ions o he inc ease in Min-C a e he imp o ed condi ions o he es ablishmen and p oli e a ion o mic obio a esponsible o he ans o ma ion and s abiliza ion o SOC p o ided by NT (La allee e al., 2020; Nunes e al., 2020). One o he ac o s ha s ongly in luences enzyme ac i i y is soil mois u e (Ade unji e al., 2017). Speci ically, in his ial, signi ican ly highe soil mois u e le els we e ound wi h he use o NT (Fe n´ andez-O ega e al., 2023a). The combina ion o highe mois u e le els and educed soil dis u bance in NT led o inc eased dehyd ogenase and β-glucosidase ac i i ies, which in u n implied g ea e SOC s abiliza ion (´ Al a o-Fuen es e al., 2013; Chen e al., 2019). Howe e , he di e ences ound ega ding Min-C we e limi ed o he su ace laye s o he soil. Min-C is ela ed wi h mine al associa ions, including chemical bonds be ween soil o ganic ma e (SOM) and min- e al su aces and occlusion wi hin mic opo es o small agg ega es (<50–63 µm) (La allee e al., 2020). I has been p o en ha NT p o- mo es he o ma ion o mic opo es and mic oagg ega es, pa icula ly in he 0–10 cm laye (G ego ich e al., 1993; Mondal and Chak abo y, 2022). On he o he hand, CT p omo es he o ma ion o mac oagg e- ga es (Talukde e al., 2023), jus i ying he lowe alues o Min-C. In deepe soil laye s, di e ences in agg ega ion a e less p onounced. I is well es ablished ha he p ima y mechanism o s abilizing ca bon in he Min-C ac ion a hese dep hs is chemical a he han physical. As a esul , he di e ences in his ac ion be ween illage sys ems end o diminish wi h inc easing soil dep h (Ti e e al., 2013). Rega ding POxC, di e ences we e only obse ed in he op 5 cm o soil due o i s g ea e mobili y (Shen e al., 2021). The highe mic obial popula ions and inc eased espi a ion ac i i y nea he soil su ace accele a e he oxida ion o compounds comp ising POxC o hei ans o ma ion in o mo e s able and p o ec ed SOC, compa ed o deepe soil laye s (Wang e al., 2017). These p ocesses a e pa icula ly obse ed when he e is an abundance o o ganic ma e inpu s (Plaza-Bonilla e al., 2014; Jagadamma e al., 2019). When analyzing he e ec s o illage a di e en dep hs on o al SOC con en , i was ound ha NT sys ems only inc eased SOC in he op 10 cm o soil. Simila ly, expe imen s conduc ed in he Medi e anean egion showed ha a e 15 yea s o expe imen a ion, NT did no signi ican ly inc ease SOC h oughou he en i e soil p o ile, bu only in he op 10 cm. In con as , CT exhibi ed he highes SOC le els when conside ing dep hs up o 40 cm (Huggins e al., 2007; ´ Al a o-Fuen es e al., 2008; Meu e e al., 2018). In ou case, a dep hs anging om 10–40 cm, NT sys ems showed simila SOC alues o CT. S udies such as hose by Blanco-Canqui and Lal (2008) ha e demons a ed ha al hough C mobili y is highe in CT sys ems han in NT, long- e m main enance o NT p omo es he ans e o ca bon o deepe soil laye s, hus elimi- na ing he di e ences be ween NT and CT o e ime (Huggins e al., 2007; Meu e e al., 2018; Zhang e al., 2018). The analysis o SOC e olu ion wi h he main enance o illage sys- ems e ealed ha while CT esul ed in a dec ease in SOC, NT allowed o he main enance o achie ed C le els. In CT sys ems, moldboa d plowing accele a es he decomposi ion o SOC and loss o C o he a - mosphe e as CO 2 . The mixing o esidues and soil h ough plowing en- hances physical con ac be ween soil mic oo ganisms and c op esidues, c ea ing mo e a o able soil mic oclima ic condi ions o esidue decomposi ion (B uce e al., 1999; Nunes e al., 2020). In con as , NT p ac ices ha e he po en ial o inc ease SOC seques a ion by educing SOC deg ada ion, lowe ing SOC mine aliza ion a es, and inc easing C inpu s (Aguile a e al., 2013; Blanco-Mou e e al., 2013; Mazzoncini e al., 2016). I is gene ally accep ed ha a e 14–15 yea s o NT p ac ices, a s a e o SOC equilib ium is achie ed, whe e no signi ican gains o losses a e obse ed in he s udied ho izons (´ Al a o-Fuen es e al., 2008; Cai e al., 2022). In ou s udy, a e 25 yea s o NT, only minimal changes in SOC we e obse ed, indica ing ha he NT ea - men ha e eached a s eady-s a e. Conside ing ha he mos signi ican losses o SOC a e obse ed in su ace ho izons (Wang e al., 2022), he adop ion o NT, which encou age C accumula ion in he su ace laye s, has p o en o be an e ec i e app oach in p ese ing SOC le els. 4.3. Ni ogen e ilize e ec s The applica ion o N e iliza ion, ega dless o he a e, inc eased SOC le els compa ed o un e ilized ea men s. This e ec ex ended o SOC ac ions, including POM-C, Min-C, and POxC. Howe e , consis en wi h indings om o he s udies conduc ed in he Medi e anean egion, highe N e iliza ion a es did no lead o addi ional inc eases in SOC le els o any o he s udied SOC ac ions (L´ opez-Bellido e al., 2010; ´ Al a o-Fuen es e al., 2013). These esul s unde sco e he bene i s o mode a e N e iliza ion a es, which enhance sys em e iciency by educing he need o excessi e mine al e ilize s. Simila o he o he ea men s examined in his expe imen , he inc eased C inpu s esul ing om e iliza ion ea men s jus i y he obse ed highe le els o POM-C (Salinas-Ga cia e al., 1997; Sainju e al., 2003; Russell e al., 2005). These indings a e consis en wi h he esul s o o he e iliza ion expe imen s (Lou e al., 2011; Lu e al., 2011; Pa eja-S´ anchez e al., 2020) and suppo he no ion p oposed by Plaza-Bonilla e al. (2014) ha POM-C is a highly esponsi e ac ion o N e iliza ion. Simila s udies ha e demons a ed ha applying app op ia e a es o N e iliza ion enhances soil mic obial communi ies (Thie elde e al., 2018; Ramí ez e al., 2020; Zhang e al., 2021). This mic obial s imu- la ion acili a es he decomposi ion o high-quali y plan li e , leading o he p oduc ion o oo exuda es and mic obial compounds (Kno e al., 2005; Co u o e al., 2013; Rocci e al., 2021). These p ocesses no only di ec ly inc ease POxC le els (Bolan e al., 2011; Ramí ez e al., 2020) bu also con ibu e o he o ma ion o Min-C h ough mecha- nisms such as di ec so p ion and he subsequen physicochemical s a- biliza ion o ca bon (Panchal e al., 2022; Lei e al., 2023). None heless, unde he condi ions o his expe imen , high a es o N e iliza ion led o an accele a ed decomposi ion o SOC, esul ing in educed SOC con en . This phenomenon is o en linked o a dec eased C: N a io, which shi s mic obial communi ies om ungal- o bac e ial- domina ed sys ems, he eby inc easing he a e o SOC decomposi ion (Six e al., 2006; Yuxin e al., 2011; Spohn e al., 2016). These indings highligh he impo ance o op imizing e iliza ion p ac ices o s ike a balance be ween main aining c op p oduc i i y and minimizing soil C losses. 5. Conclusion In Medi e anean soils, i iga ed maize monoc opping unde con- en ional illage has been shown o dec ease SOC le els due o in en- si ied o ganic ma e decomposi ion and insu icien ca bon inpu s. T ansi ioning o legume-maize double-c opping sys ems has p o en o be use ul in main aining SOC le els by inc easing C inpu s and imp o ing SOC cycling. Simila ly, adop ing no- illage p ac ices im- p o es SOC seques a ion in su ace laye s h ough educed dis u bance and enhanced biological ac i i y linked o ca bon s abiliza ion. While ni ogen e iliza ion suppo s c op p oduc i i y and con ibu es o ca bon inpu s, excessi e a es accele a e SOC decomposi ion, high- ligh ing he need o op imized applica ion a es o balance ca bon e en ion and sys em e iciency. In he ace o ising empe a u es and clima ic challenges in Medi- e anean egions, in eg a ing legume-maize double-c opping, no- illage, and mode a e ni ogen e iliza ion eme ges as a sus ainable s a egy o main ain SOC le els, enhance mic obial unc ion, and J. Fe n´ andez-O ega e al. Soil & Tillage Resea ch 249 (2025) 106496 8 imp o e soil esilience. CRediT au ho ship con ibu ion s a emen Delgado An onio: Me hodology, In es iga ion. Ga cía-L´ opez Ana: Me hodology, In es iga ion. Fe n´ andez-O ega Jesús: W i ing – o ig- inal d a , Visualiza ion, So wa e, Me hodology, In es iga ion, Fo mal analysis, Da a cu a ion. Can e o-Ma ínez Ca los: Supe ision, In es- iga ion, Funding acquisi ion. ´ Al a o-Fuen es Jo ge: W i ing – e iew & edi ing, Supe ision, Funding acquisi ion. Decla a ion o Compe ing In e es The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Acknowledgemen s The au ho s would like o hank Ca los Co ´ es and Sil ia Ma í o labo a o y and ield assis ance. This esea ch wo k was inancially suppo ed by he Minis e io de Ciencia e Inno aci´ on o Spain (p ojec AGL2017–84529-C3–3-R;; PhD ellowship PRE2018–084610). Da a a ailabili y Da a will be made a ailable on eques . Re e ences Ade unji, A.T., Lewu, F.B., Mulidzi, R., Ncube, B., 2017. The biological ac i i ies o β-glucosidase, phospha ase and u ease as soil quali y indica o s: a e iew. J. Soil Sci. Plan Nu . 17, 794–807. Aguile a, E., Lassale a, L., Ga inge , A., Gimeno, B.S., 2013. Managing soil ca bon o clima e change mi iga ion and adap a ion in Medi e anean c opping sys ems: a me a-analysis. Ag ic. Ecosys . En i on. 168, 25–36. Ajwa, H.A., Taba abai, M.A., 1994. Decomposi ion o di e en o ganic ma e ials in soils. Biol. Fe il. Soils 18, 175–182. Allen, R.G., Pe ei a, L.S., Raes, D., & Smi h, M., 1998. 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