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Toma o g ow h and physiology as well as soil physicochemical and biological
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p ope ies a ec ed by ozona ed wa e in a saline ag oecosys em
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Ma a Díaz-López1,2 *, Lucas Gale a3 Felipe Bas ida2, Emilio Nicolás1
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*co esponding au ho ([email p o ec ed])
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1Depa men o I iga ion, CEBAS-CSIC, Campus Uni e si a io de Espina do, Mu cia,
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30100, Spain.
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2Depa men o Soil and Wa e Conse a ion and Was e Managemen , CEBAS-CSIC,
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Campus Uni e si a io de Espina do, Mu cia, 30100, Spain.
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3No ag ic (No edades Ag ícolas, S.A.), Bule a de Vica 743, 04738 Vica , Alme ía,
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Spain
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Manusc ip (double-spaced and con inuously LINE and PAGE
numbe ed)- o inal publica ion
Click he e o iew linked Re e ences
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Abs ac
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Cu en ends in ag icul u e a e ocused on implemen ing sus ainable p ac ices ha
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a oid he use o chemical compounds. In his con ex , i iga ion wi h ozona ed wa e
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could be a po en ial s a egy o educe some chemical compounds in soils due o he
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deg ada i e powe o ozone. Howe e , he e ec s o i iga ion wi h ozona ed wa e on
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he soil mic obial communi y and plan ag ophysiology a he ield scale a e la gely
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unknown. He e, we s udied he impac o i iga ion wi h ozona ed wa e on he mic obial
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communi y o a Medi e anean soil, and on Solanum lycope sicum L. ag o-physiology
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and p oduc i i y in a g eenhouse expe imen . To his end, we e alua ed: i) soil
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physicochemical p ope ies, soil enzyme ac i i ies, mic obial biomass ia a y acid
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analysis, mic obial di e si y ( ia amplicon sequencing), and ii) he nu ien con en ,
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physiology, phy oho mone con en , yield, and ui quali y o oma o plan s. Ou esul s
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indica e ha soil physicochemical p ope ies we e signi ican ly a ec ed by he i iga ion
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wi h ozona ed wa e (OZ). We obse ed an inc ease in he con en o o al o ganic ca bon
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(TOC), wa e -soluble ni ogen (WSN) and ammonium, and a dec ease in soil pH due o
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he OZ ea men . In addi ion, a signi ican inc ease in alkaline phospha ase and ungal
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and bac e ial biomass was also obse ed in he OZ ea men . I was obse ed ha he
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p oka yo ic communi y s uc u e was a ec ed by he OZ ea men , while ha o ungi
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was undis u bed. The OZ ea men inc eased he pho osyn he ic a es o oma o plan s
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and main ained wa e condi ions when compa ed o con ol plan s. The inc eased ans-
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Zea in iboside ( Z-Rib) could p o ide apid apical and oo g ow h allowing adap a ion
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o he new g owing condi ions. Howe e , a mo e in-dep h s udy on he physiological
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esponse o he plan o his ea men would be o in e es , as i would help wi h he
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implemen a ion o his s a egy in ag icul u al ields in a sa e manne , and wi h ob aining
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highe plan yields.
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Keywo ds: Soil mic obial communi y; Ozone; Enzyme ac i i y; Mic obial biomass;
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Plan physiology; Phy oho mone; Toma o.
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1. In oduc ion
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Cu en ends in ag icul u e ocus on applying sus ainable p ac ices ha a oid he
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use o chemical compounds such as pes icides, ungicides, e c. Some o hese s a egies
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include i) bio-based sus ainable in ensi ica ion, ii) ag oecological ans o ma ion, and iii)
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polycul u es and c op o a ion (FAO, 2019). In his con ex , ozona ion o i iga ion wa e
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could also be a po en ial s a egy o educe he con en o some o hese chemical
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compounds in soils. Some s udies ha e ocused on his issue, bu ha e paid less a en ion
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on he e ec s o ozona ed wa e on he soil mic obial communi y, which plays a c i ical
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ole in soil e ili y, and plan ag o-physiological pa ame e s (Ding e al., 2018; Ghah chi
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& Rezaee, 2020). Thus, i is impo an o e alua e he e ec s o ozona ed wa e on soil
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e ili y and plan ag o-physiology be o e implemen ing his s a egy on a la ge
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ag icul u al scale. Ne e heless, he e is li le in o ma ion o he e ec s on c ops o ozone
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applied o he i iga ion wa e .
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Some au ho s ha e poin ed ou ha i iga ion wi h ozona ed wa e can p omo e he
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accumula ion o seconda y me aboli es, he ac i a ion o he an ioxidan appa a us, and
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he o e p oduc ion o phy oho mones (Risoli and Lau ia, 2022). Consequen ly, a highe
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esis ance agains pa hogen in ec ion can be p omo ed (Landa Fe nández e al., 2019;
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P igigallo e al., 2019). Thus, i iga ion wi h ozona ed wa e allows oxygena ion in he
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plan oo a ea, and can help o sani ize i iga ion sys ems, especially o p e en he sp ead
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o pa hogens (Zheng e al., 2020). Howe e , when p esen in aqueous solu ions, ozone
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unde goes apid b eakdown in o molecula oxygen and di e en eac i e species o
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oxygen (ROS), as i is an uns able compound ( on Sonn ag & on Gun en, 2015). The
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inc ease in he concen a ion o ROS in he i iga ion wa e can cause oxida i e s ess on
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plan s and ul ima ely a ec ing hei g ow h and de elopmen (Zheng e al., 2020). In
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compa ison o i s e ec on plan s, li le is known abou he e ec s o ozona ed wa e
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applica ion on soils. Moni o ing he e ec s o ag icul u al p ac ices in he soil mic obial
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communi y is undamen al, gi en he impo an ole o soil mic obes in he main enance
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o soil e ili y and ecosys em se ices (Delgado-Baque izo e al., 2016). Indeed,
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al e a ions in he composi ion and unc ionali y o he soil mic obial communi y can lead
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o loss o soil heal h, esul ing in he deg ada ion o he soil o ag icul u al use, and
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con ibu ing o soil deg ada ion and e osion (Use o e al., 2021). Se e al app oaches ha e
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been widely used o ack he esponses o he soil mic obial communi y o ag icul u al
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managemen , om he measu emen o soil enzyme ac i i ies ela ed o nu ien cycles
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(Lehmann e al., 2020) o he composi ion and di e si y o he bac e ial and ungal
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communi ies by amplicon sequencing.
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This wo k aims o e alua e he e ec o i iga ion wi h ozona ed wa e on he
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pe o mance o Solanum lycope sicum ( oma o) and on he mic obial communi y, o a
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semia id Medi e anean soil unde salini y condi ions. In pa icula , we expec changes in
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he di e si y and composi ion o he mic obial communi y, bu a educed impac in
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ex acellula soil enzyme ac i i ies, which a e usually p o ec ed in soil mine als and
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o ganic ma e (Bu ns e al., 2013). Mo eo e , we expec ha i iga ion wi h ozona ed
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wa e would inc ease he a ailabili y o nu ien s in he soil, and consequen ly modi y
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bo h plan physiology and yield (Ikeu a e al., 2018). I iga ion wi h ozona ed wa e is
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expec ed o in luence se e al physiological pa ame e s such as ne pho osyn hesis,
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s oma al conduc ance, and phy oho mone con en (Tahamolkonan e al., 2022). In
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pa icula , he inc ease in salicylic acid con en has been epo ed (P igigallo e al. 2019),
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which could dec ease he s oma al conduc ance. Mo eo e , simila le els o ne
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pho osyn hesis (Ma ínez-Sánchez & Aguayo, 2019) can lead o highe le els o in insic
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wa e use e iciency. Fo his pu pose, we e alua ed i) he soil mic obial biomass,
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composi ion, and enzyme ac i i ies, oge he wi h soil chemical pa ame e s and nu ien
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con en s, and ii) he c op nu ien con en , physiology, phy oho mone con en , yield, and
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ui quali y. We hypo hesized ha changes in soil physico-chemical pa ame e s (e.g.
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educ ion in soil pH, inc ease in elec ical conduc i i y, e c.) upon ozone i iga ion, will
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a ec he a ailabili y o nu ien s and he e o e he soil mic obial communi y and plan s.
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In addi ion, we expec sligh e ec o i iga ion wi h ozona ed wa e on he plan nu i ion
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bu some signi ican e ec on physiological pa ame e s due o he inc ease in oxida i e
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molecules.
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2. Ma e ials and me hods
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2.1. Expe imen al design
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The expe imen was conduc ed in a g eenhouse loca ed in Mu cia, sou heas e n Spain
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(37º28’N 1º32’W) du ing Augus -May 2020-2021. The selec ed soil was a sandy-loam
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soil (14.7% clay, 21.3% sil , 64% sand) wi h he ollowing cha ac e is ics: pH (H2O) 7.57
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± 0.20; elec ical conduc i i y (dS m−1) 8.44 ± 3.28; o ganic C con en (%) 1.89 ± 0.25;
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o al N (%) 0.39 ± 0.09; alkalini y (% CaCO3) 2.68 ± 1.59. The soil p esen ed a high
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concen a ion o Na, eaching 21% o he exchangeable sodium pe cen age, and can
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he e o e be conside ed a saline-sodic soil.
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The ea men s e alua ed in his s udy we e: 1) d ip i iga ion wi hou ozona ed wa e
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(C), and 2) d ip i iga ion wi h ozona ed wa e (OZ). The i iga ion wa e was collec ed
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and il e ed using se e al p ocedu es: i) mul i-laye ed sand bed il a ion, ii) disc sys em
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il a ion, and iii) ul a il a ion memb anes il a ion. The wa e ozona ion was pe o med
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in si u using comme cial equipmen p o ided by NOVAGRIC, S.A (u ili y model
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ES1256014). Ozone, wi h a edox po en ial o 800-850mV, was gene a ed om
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a mosphe ic ai and inally injec ed in o he il e ed i iga ion wa e (Table S1). The
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ea men s we e andomly a anged and dis ibu ed in he g eenhouse (900 m2), esul ing
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in ou eplica es pe ea men . Each eplica e consis ed o 120 Solanum lycope sicum
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plan s dis ibu ed in eigh ows, wi h a dis ance o 1 m be ween ows and 40 cm be ween
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plan s in he same ow (2.5 plan s m−2). The plan s we e i iga ed wi h d ippe s, wi h one
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p essu e-compensa ed emi e pe plan discha ging 2 L h-1, which esul ed in an i iga ion
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o 2000 m3 ha-1 o wa e du ing he o al g owing season. In o de o ensu e he co ec
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de elopmen o he plan s, all o hem ecei ed he same amoun o he main
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mac onu ien s (N-P2O5-K2O): 240-105-405, and seconda y mac onu ien s: 44 CaO and
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26 MgO (kg ha-1), h ough he d ip i iga ion sys em.
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The soil samples we e collec ed as ollows: h ee soil samples we e aken (0–15 cm
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o dep h) and mixed o ob ain one composi e sample pe eplica e. Each soil eplica e was
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sampled in he middle o one ow o oma o plan s. Soil samples we e aken a h ee
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di e en imes: Decembe -2020 ( 1), Feb ua y-2021 ( 2), and May-2021 ( 3),
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co esponding o he beginning o ha es ( 1), middle o ha es ( 2), and end o ha es
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( 3) pe iods. The samples we e sie ed (2 mm) and kep a 4 °C o chemical analyses and
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a -20 °C o a y acid me hyl es e analysis and DNA ex ac ion. All he soil and plan
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analyses we e pe o med om he middle ow o each eplica e in o de o a oid he edge
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e ec . Fi e plan s om each eplica e (2.5 plan s m−2) we e chosen o physiological and
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nu ien con en analyses. The p oduc i i y and quali y o he ui s we e also e alua ed
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in hese plan s. Gas exchange pa ame e s, chlo ophyll con en , and phy oho mone
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de e mina ion we e pe o med a he same ime as 2 soil sampling. A his ime, he
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oma o plan s we e in ull ui p oduc ion.
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2.2. Soil physicochemical pa ame e s, enzyme ac i i ies, and a y acid me hyl es e
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(FAME) analysis
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The pH and elec ical conduc i i y (EC) o he soil we e measu ed using a C ison
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GLP 21 pH-me e and a C ison CM 2200 conduc i i y-me e (C ison Hach Lange, Alella,
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Spain), espec i ely, om a soil:wa e ex ac (1:5, w: ). The o al N (TN), o al C (TC),
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and o al o ganic C (TOC) o he soil we e analyzed wi h an Elemen al Analyze (C/N
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Flash EA 112 Se ies-Leco T uspec). The soil wa e -soluble C (WSC) and wa e -soluble
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N (WSN) we e de e mined by an analyze o liquid samples (Mul i N/C 3100, Analy ic
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Jena, Ge many) om a soil:wa e ex ac (1:5, w: ). The de e mina ion o he soil
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ammonium con en and he u ease ac i i y we e analyzed by he Kandele and Ge be
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(1988) me hod. The alkaline phosphomonoes e ase and β-glucosidase ac i i ies we e
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analyzed wi h he me hod by Taba abai and B emne (1969), and he Ei azi and Taba abai
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(1988) me hod, espec i ely.
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Fa y acid me hyl es e s (FAMEs), he ea e a y acids, we e ex ac ed om 3 g o
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soil acco ding o Schu e and Dick (2000), and we e used as indica o s o he soil
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mic obial biomass. The a y acids ep esen a i e o he bac e ial biomass we e i15:0,
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a15:0, i16:0, i17:0, 16:1ω7, cy17:0, cy19:0, 10Me16:0, and 10Me18:0 (Dungai e al.,
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2011; F os egå d e al., 1993), and he a y acids indica o s o he ungal biomass we e
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18:2ω6,9 and 18:2ω6,9c (B an e al., 2006; Rinnan & Båå h, 2009). The ela i e
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abundances o all he a y acids iden i ied and he ungal: bac e ial a y acids a io we e
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used o he analysis o he changes in he s uc u e o he mic obial communi y.
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2.3. DNA ex ac ion, amplicon sequencing, and indica o gene a o he p oka yo ic
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communi y
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Soil samples om 2 and 3 we e selec ed o u he explo e he di e si y and
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composi ion o hei bac e ial and ungal communi ies by amplicon sequencing. The
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amplicon sequencing p o ided us wi h a deepe unde s anding o he s a us o he soil
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p oka yo ic and ungal communi ies a a pa icula ime. These soil sampling imes we e
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chosen because hey co esponded o he middle o ha es ( 2) and he end o ha es ( 3)
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pe iods o oma o plan s. DNA ex ac ion was pe o med om 250 mg o soil using he
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DNeasy Powe Soil P o Ki (Qiagen). The 515F and 806R p ime pai om (Capo aso e
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al., 2012) was used o ampli y he V4 egion o he p oka yo ic 16S RNA gene, and he
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gITS7 and ITS4 pai (Ih ma k e al., 2012) o he ampli ica ion o he ungal ITS2 egion.
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The PCR condi ions and sequencing p ocedu e we e as desc ibed in Díaz-López e al.
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(2021).
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The sequences we e p ocessed using he USEARCH pipeline and he UPARSE-OTU
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algo i hm (Edga , 2013). Fi s , aw MiSeq pai ed-end eads om he 16S RNA gene and
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he ITS2 egion we e assembled sepa a ely. Then, he sequences we e quali y- il e ed,
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allowing a maximum e- alue o 0.5 o he 16S lib a y and 1.0 o he ITS2 lib a y,
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B e ibacillus, Mic obispo a, Idioma ina, and Tumebacillus we e signi ican ly educed
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by his ea men (p < 0.05).
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3.4. Gas exchange pa ame e s, chlo ophyll con en , and phy oho mone con en
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The gas exchange pa ame e s, chlo ophyll con en , and phy oho mone con en o
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oma o lea es (Table 2) we e measu ed in o de o de e mine he esponse o he oma o
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plan s o he ozona ed wa e ea men . All hese analyses we e conduc ed a 2, a he
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same ime ha soil was sampled, coinciding wi h he middle o he ha es pe iod. The
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esul s ob ained showed a signi ican (p < 0.05) inc ease in he ne pho osyn hesis (A)
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and he s oma al conduc ance (gs) in he plan s i iga ed wi h ozona ed wa e . Howe e ,
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we did no obse e signi ican di e ences in he in insic wa e use e iciency (iWUE).
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The inc ease in A was no coupled wi h a highe chlo ophyll con en , ei he chlo ophyll
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a (Chl a), b (Chl b), o o al (Chl T) con en . None heless, he e was a end o he
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ozona ed wa e ea men plan s o ha e a g ea e chlo ophyll con en .
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In o de o u he s udy he physiological esponse o oma o plan s o he ozona ed
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wa e ea men , we e alua ed he phy oho mone con en (Table 3) a he same ime as
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we e alua ed he gas exchange pa ame e s. The OZ ea men signi ican ly inc eased (p
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< 0.05) he ans-Zea in iboside ( Z-Rib) con en . Howe e , in he same ea men , a
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signi ican dec ease (p < 0.05) was obse ed in gibbe ellic acid 7 (GA7), abscisic acid
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(ABA), and salicylic acid (SA) con en .
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3.5. Lea mine al con en , c op yield, and ui quali y
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The nu i ional s a us o oma o plan s was analyzed a h ee di e en imes
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h oughou he ha es pe iod. We did no ind signi ican di e ences in he elemen s
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analyzed (Table S2), indica ing ha he ozona ed wa e ea men did no in luence he
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nu i ional s a us o he oma o plan s unde hese s udy condi ions. We also e alua ed he
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o al yield and he ui quali y h ough he whole ha es pe iod. No signi ican
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di e ences we e ound in yield o ui quali y pa ame e s (Table S3). Las ly, we analyzed
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he cumula i e yield and did no obse e any signi ican di e ences be ween ea men s
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ei he (Figu e 6).
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4. Discussion
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4.1. Soil chemical, biochemical and mic obial esponses o ozona ed wa e
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The e alua ion o soil physicochemical and biochemical cha ac e is ics is o g ea
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impo ance, as some o hem a e di ec ly ela ed o soil quali y and heal h. When applied
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o i iga ion wa e , ozone apidly decomposes in o molecula oxygen and HO adicals
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( on Sonn ag & on Gun en, 2015). As a esul , HO adicals eac wi h he labile
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compounds p esen in he soil and elease p o ons (H+), sal s, and o he compounds in o
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he soil (Ghah chi & Rezaee, 2020), which could explain he signi ican educ ion in soil
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pH wi h he OZ ea men . Howe e , elec ical conduc i i y was no a ec ed by he OZ
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ea men , possibly due o he high alues obse ed in he ini ial measu emen o he soil.
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Main aining o inc easing he C and N con en o he soil is key in u u e ag icul u al
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s a egies (Lekbe g e al., 2021). In ou case, he OZ ea men showed a signi ican
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inc ease in TOC and WSN con en , especially in ammonium con en . S udies ha e shown
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ha he adicals eleased by he decomposi ion o ozone in i iga ion wa e could ha e
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enhanced he decomposi ion o o ganic ma e and he gene a ion o labile compounds in
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he soil (Díaz-López e al. 2021; Wang and Chen 2020; Yu e al. 2005). Mo eo e , he
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inc ease in TOC and WSN compounds could be due o he combina ion o se e al ac o s
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ha sepa a ely may no be signi ican bu ha inally a ec in a signi ican way. Fo
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example, inc eased ROS con en could ha e led o inc eased exuda e p oduc ion by plan s
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(Risoli & Lau ia, 2022), and on he o he hand, he plan may abso b ewe nu ien s om
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he soil and cause hem o accumula e o a g ea e ex en han in he non-ozona ed wa e
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plan s. Fu he , he e a e e idences ha oxygena ing he oo a ea, which may also occu
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he e h ough quick ozone decomposi ion, can inc ease oo g ow h and o ganic C in soil
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(Zhou e al., 2022).
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Soil enzymes and hei ac i i ies ca alyze many nu ien cycles in soils, and a e
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commonly used as indica o s o soil quali y and e ili y (Bowles e al., 2014; Bu ns e
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al., 2013). The enzyme ac i i ies es ed (β-glucosidase, u ease, and alkaline
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phosphomonoes e ase) in his s udy we e main ained o e en inc eased wi h he OZ
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ea men . The signi ican inc ease in soil phosphomonoes e ase ac i i y in he OZ
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ea men may indica e a g ea e enzyme p oduc ion by he soil mic obial biomass, la ge
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subs a e a ailabili y, and/o g ea e phospho us demand by oma o plan s. The ac i i y
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o his enzyme is key o he phospho us cycle, wi h his mac onu ien being commonly
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limi ed in soils. The obse ed enzyme ac i i ies co ela ed wi h he mic obial biomass, as
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es ima ed by a y acids, which ein o ce he idea ha hese enzymes p oduce easily
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a ailable subs a es o bo h plan and mic obial g ow h.
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Mic obial a y acids p o ide an es ima ion o soil mic obial biomass (Fanin e al.,
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2019), and can be use ul o moni o ing he po en ial e ec s o ozone, which has
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ecognized disin ec an ole (Mi sugi e al., 2014), in he soil mic obial communi y.
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O e all, ou esul s indica ed ha ozone did no nega i ely impac soil mic obial biomass,
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and his migh be likely due o: i) he g ea e a ailabili y o nu ien s as a esul o he
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highe deg ada ion eac ions ca ied ou by he ozone- eleased adicals (Wang e al.,
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2019), ii) he educ ion o he an imic obial e ec o ozone due o i s quick b eakdown in
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wa e , and iii) he inc ease o soil and hizosphe e ae a ion due o he elease o O2 on
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decomposi ion o O3 in wa e (Zhou e al., 2022).
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A deepe insigh h ough amplicon sequencing analysis would shed ligh on he
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e ec s o ozone applica ion in i iga ion wa e and how i a ec s soil mic obial
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communi ies (Use o e al., 2021). P e ious s udies obse ed a negligible e ec o
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i iga ion wi h ozona ed wa e on he s uc u e o he soil mic obial communi y (Díaz-
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López e al., 2021). Howe e , in his assay, we ha e e alua ed a longe pe iod o ime,
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p o ided a mo e ealis ic e idence o he long- e m e ec s o he applica ion o he
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ea men unde s udy. In his ega d, he esul s o he NMDS analysis indica ed ha he
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p oka yo e communi y was signi ican ly a ec ed by he ea men s analyzed, ega dless
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o he sampling ime. The ungal communi y, on he con a y, was no a ec ed ei he by
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ime o by ea men . These esul s a e con adic o y o hose obse ed in p e ious ials,
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in which simila g owing condi ions we e e alua ed (Díaz-López e al., 2021). Howe e ,
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i should be no ed ha he cha ac e is ics o he soils s udied we e e y di e en , so he
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communi ies ha inhabi hese soils also ha e di e en cha ac e is ics and po en ial
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esponses o ozona ed wa e . The soil in he p esen s udy had a high salini y, which
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u he limi s he abili y o mic oo ganisms o adap o new ield condi ions imposed by
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ozona ed wa e (Wang and Bao 2022). When analyzing he ela i e abundance o
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p oka yo ic o de s, we obse ed ha he e we e small di e ences be ween ea men s in
394
20
he communi y composi ion. Howe e , by s udying genus indica o s, we can disco e
395
which gene a a e be e adap ed o he speci ic condi ions c ea ed by he i iga ion wi h
396
ozona ed wa e . The OZ ea men , ega dless o he sampling ime, p esen ed 13 genus
397
indica o s, wi h some o hem being bac e ia wi h salini y ole ance, as G acillibacillus,
398
Noca diopsis, P ause ella, and Salinibacillus. Howe e , li le is known abou hese
399
gene a and he po en ial impac hey may ha e on bo h soil e ili y and c op p oduc i i y.
400
Fu he in es iga ion is needed o be e unde s and he long- e m e ec s o i iga ion
401
wi h ozona ed wa e .
402
4.2. Plan physiology and p oduc i i y unde i iga ion wi h ozona ed wa e
403
I is well known ha soil-plan in e ac ion occu s, so ha changes o al e a ions in
404
soil condi ions can a ec plan ’s physiology (Sasse e al., 2018; Zak e al., 2003). As a
405
esul , plan de elopmen can be posi i ely o nega i ely modula ed by changes in he
406
soil en i onmen . I is he e o e essen ial o join ly e alua e he soil-plan ela ionship
407
when implemen ing new s a egies ocused on a mo e sus ainable ag icul u e. Mo eo e ,
408
in comme cial c ops, i is e y impo an o e alua e he ag onomic pa ame e s as well as
409
he plan ’s physiology, since he main pu pose is o look o a g ea e p oduc i i y wi hou
410
a ec ing ui quali y.
411
P e ious s udies ha e obse ed imbalances in he nu i ional con en o plan s in
412
c ops g own wi h ozona ed nu ien solu ion (Bou Jaoudé e al., 2008). This is mainly due
413
o he p ecipi a ion o some elemen s in he nu ien solu ion (Ikeu a e al., 2018), making
414
hem inaccessible o he plan . Howe e , in ou case, we did no ind any al e a ions in
415
he nu ien con en o he plan s. The decomposi ion o ozone, in ac , a o s he elease
416
21
o labile compounds ha a e linked o he o ganic ma e in he soil (Ghah chi & Rezaee,
417
2020; Rizzo e al., 2020). Mo eo e , he inc ease in soil phosphomonoes e ase ac i i y
418
a o s he elease o simple P o ms o plan s. In his way, he oxida i e s ess ha can
419
be po en ially gene a ed by i s ee adicals could be a enua ed by he posi i e e ec s o
420
i iga ion wi h ozona ed wa e a he nu i ional le el.
421
Ozone gas exposu e has been shown o damage pho osyn he ic p ocesses (Caille e
422
e al., 2018). Howe e , he OZ ea men induced an inc ease in ne pho osyn hesis (A)
423
and s oma al conduc ance (gs), leading o an in insic wa e use e iciency (iWUE) simila
424
o he con ol. In ac , an inc eased plan chlo ophyll con en was obse ed in he OZ
425
ea men . This could indica e ha highe pho osyn he ic a es and be e wa e condi ions
426
can be achie ed wi h he OZ ea men . In addi ion, c op yield is closely ela ed o he
427
physiological and nu i ional s a us o he plan . Howe e , he e is no gene al ule o
428
oma o c ops, as yield a ies depending on many en i onmen al and a ie al ac o s and
429
he ag icul u al p ac ices adop ed. In ou case, he oma o plan s had a simila p oduc i i y
430
wi h o wi hou i iga ion wi h ozona ed wa e . Also, i iga ion wi h ozona ed wa e did
431
no signi ican ly al e he p oduc i i y o oma o plan s and ui quali y.
432
Del ing deepe in o he plan ’s esponses o ozone, we in es iga ed he ho mone
433
pa e n. (P igigallo e al., 2019; Risoli & Lau ia, 2022). I iga ion wi h ozona ed wa e
434
sligh ly a ec ed he phy oho mone con en in his assay. The inc ease in s oma al
435
conduc ance could be explained by he educ ion in ABA and SA con en in he lea es
436
(Wilkinson & Da ies, 2010). In con as , Z-Rib con en inc eased wi h he OZ ea men .
437
The physiological ole o Z-Rib is o con ol lea size, as well as ai s ela ed o me is em
438

22
ac i i y, whe eas Z can only con ol lea size (Osugi e al., 2017). The e o e, apical and
439
oo g ow h can be quickly modula ed by Z-Rib, allowing he apid adap a ion o new
440
g owing condi ions. Howe e , he physiological e ec o i iga ion wi h ozona ed wa e
441
on c ops mus be s udied u he , o adequa ely implemen his s a egy in ag icul u al
442
ields.
443
5. Conclusions
444
I iga ion wi h ozona ed wa e has been shown o ha e an in luence on soil p ope ies
445
and oma o physiology. A he soil le el, he inc ease in bac e ial biomass and in some o
446
he enzyme ac i i ies analyzed (e.g., alkaline phosphomonoes e ase) could indica e ha
447
i iga ion wi h ozona ed wa e has posi i e e ec in he soil mic obial communi y, which
448
is impo an ly linked o soil heal h and e ili y, and also could imp o e plan
449
de elopmen . This could be due o i) inc eased nu ien a ailabili y, ii) a educ ion in he
450
an imic obial e ec o ozone due o i s apid decomposi ion in wa e , and iii) inc eased
451
soil and hizosphe e ae a ion. A he plan le el, i iga ion wi h ozona ed wa e seems o
452
modula e he plan ’s esponse a he physiological le el, while a he ag onomic and
453
nu i ional le els, he e we e no signi ican changes. The OZ ea men inc eased he
454
pho osyn he ic a es and main ained wa e condi ions as compa ed o he con ol. In
455
addi ion, inc eased Z-Rib could p o ide apid apical and oo g ow h allowing o a apid
456
adap a ion o he new g owing condi ions. Howe e , a mo e in-dep h s udy on he
457
physiological esponse o he plan o his ea men would be o in e es , as i would help
458
wi h he implemen a ion o his s a egy in ag icul u al ields in a sa e manne , and wi h
459
23
ob aining highe plan yields. Ou esul s sugges ha soil e ili y and oma o plan
460
p oduc i i y we e no signi ican ly comp omised by i iga ion wi h ozona ed wa e .
461
462
Acknowledgmen s
463
The au ho s acknowledge Ma io G. Fon o his co ec ions and sugges ions o he
464
English g amma in his manusc ip .
465
Funding
466
This wo k was suppo ed by he Eu opean Commission h ough he LIFE+ P og am
467
(LIFE17 ENV/ES/000203 - LIFE AGREMSO3IL). The au ho s a e also g a e ul o he
468
Fundación Séneca (19903/GERM/15 and 19896/GERM/15), he p ojec PID2020-
469
114942RB-I00 unded by MCIN/AEI/10.13039/501100011033, and he p ojec
470
PID2019–106226RB-C21/AEI/10.13039/501100011033). This s udy o med pa o he
471
AGROALNEXT p og amme and was suppo ed by MCIN wi h unding om Eu opean
472
Union Nex Gene a ionEU (PRTR-C17.I1) and by Fundación Séneca wi h unding om
473
Comunidad Au ónoma Región de Mu cia (CARM).
474
Decla a ions
475
Compe ing in e es s. The au ho s decla e no compe ing in e es s.
476
Supplemen a y in o ma ion
477
Supplemen a y da a o his a icle can be ound online a
478
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130050. h ps://doi.o g/10.1016/j.jclep o.2021.130050
672

33
673
674
34
Figu e cap ions:
675
Figu e 1. Physicochemical and chemical p ope ies o he s udied soils: C (con ol), OZ
676
(i iga ion wi h ozona ed wa e ). Time: 1 (Decembe ), 2 (Feb ua y), and 3 (May). EC
677
(elec ical conduc i i y), TOC ( o al o ganic C) and WSN (wa e -soluble ni ogen). Ba s
678
ep esen he mean and he e o ba s ep esen he SD. Fo each ime poin , da a ollowed
679
by di e en le e s a e signi ican ly di e en (p <0.05).
680
Figu e 2. Enzyme ac i i ies in he s udied soils C (con ol), OZ (i iga ion wi h ozona ed
681
wa e ). Time: 1 (Decembe ), 2 (Feb ua y), and 3 (May). Ba s ep esen he mean and
682
he e o ba s ep esen he SD. Fo each ime poin , da a ollowed by di e en le e s a e
683
signi ican ly di e en (p <0.05).
684
Figu e 3. Fa y acid con en s ep esen a i e o di e en mic obial g oups, and he a ios
685
be ween mic obial g oups, in he s udied soils: C (con ol), OZ (i iga ion wi h ozona ed
686
wa e ). Time: 1 (Decembe ), 2 (Feb ua y), and 3 (May). Ba s ep esen he mean and
687
he e o ba s ep esen he SD. Fo each ime poin , da a ollowed by di e en le e s a e
688
signi ican ly di e en (p <0.05).
689
Figu e 4. Non-me ic dimensional scaling (NMDS) biplo o he B ay-Cu is
690
dissimila i y ma ix o he p oka yo ic (A) and ungal (B) communi y composi ions. The
691
PERMANOVA P alues a e 0.0038 and 0.4107 o p oka yo es and ungi, espec i ely.
692
C-T2: con ol a 2; OZ-T2: i iga ion wi h ozona ed wa e a 2; C-T3: con ol a 3; OZ-
693
T3: i iga ion wi h ozona ed wa e a 3. Time: 2 (Feb ua y), and 3 (May).
694
Figu e 5. Rela i e abundances o p oka yo ic phyla (A) and ungal (B) o de s in he
695
s udied soils wi h abundance > 1% in a leas one ea men . C-T2: con ol a 2; OZ-T2:
696
35
i iga ion wi h ozona ed wa e a 2; C-T3: con ol a 3; OZ-T3: i iga ion wi h ozona ed
697
wa e a 3. Time: 2 (Feb ua y), and 3 (May).
698
Figu e 6. Cumula i e yield o oma o ui s du ing he ha es pe iod (Dec o May). C
699
(con ol), OZ (i iga ion wi h ozona ed wa e ).
700
701
Tables
Table 1. P oka yo ic genus indica o o ea men s and sampling imes analyzed, and
also ea men s ega dless o sampling ime (p <0.05). Sampling ime: 2 (Feb ua y) and
3 (May).
T ea men s
Genus indica o
Con ol 2
Ozone 2
Cy obacillus
Isop e icola
Oceanobacillus
S ep omonospo a
Con ol 3
Halomonas
Idioma ina
Salinimic obium
Ozone 3
Con ol ( 2 + 3)
Caldo a us
Mic obispo a
Pedomic obium
Pseudog acilibacillus
The momonospo a
Tumebacillus
Ozone ( 2 + 3)
Alii odinibius
Flinde siella
G acilibacillus
Jiangella
Mycobac e ium
Noca diopsis
Phy oac inopolyspo a
P ause ella
Saccha omonospo a
Saccha opolyspo a
Salinibacillus
S ep omonospo a
T uepe a
Table 2. Gas exchange pa ame e s and chlo ophyll con en a 2 (Feb ua y). Values
ep esen he mean and he SD (pa en hesis). Da a ollowed by di e en le e s a e
signi ican ly di e en (p <0.05).
Table (Edi able e sion) Click he e o access/download;Table (Edi able
e sion);Tables_Diaz-Lopez2022.docx
Gas exchange
Con ol
Ozone
A (µmol CO2 m−2 s−1)
11.77 (3.55) b
20.27 (1.67) a
gs (mol H2O m−2 s−1)
0.09 (0.03) b
0.17 (0.02) a
iWUE (µmol CO2 mol H2O-1)
135.83 (25.18)
119.04 (13.06)
Chlo ophyll con en
Chl a (mg g-1)
1.49 (0.29)
1.70 (0.17)
Chl b (mg g-1)
0.47 (0.10)
0.55 (0.06)
ChlT (mg g-1)
1.96 (0.38)
2.25 (0.23)
A: ne pho osyn hesis; gs: s oma al conduc ance; iWUE: in insic wa e use e iciency.
Table 3. Phy oho mone con en (ng g-1) a 2 (Feb ua y). Values ep esen he mean and
he SD (pa en hesis). Da a ollowed by di e en le e s a e signi ican ly di e en (p
<0.05)
Phy oho mone con en
Con ol
Ozone
ACC
34.86 (6.46)
30.84 (5.82)
Z
72.21 (13.08)
62.29 (12.25)
Z-Rib
1.14 (0.45) b
2.35 (0.64) a
GA4
2.19 (1.17)
2.18 (1.55)
GA7
95.88 (16.51) a
38.23 (11.51) b
ABA
39.35 (4.10) a
28.91 (3.02) b
SA
46.60 (14.22) a
15.91 (3.08) b
ACC: 1-aminocyclop opane-1-ca boxylic acid; Z: ans-Zea in; Z-Rib: ans-Zea in iboside;
GA4: gibe ellic acid; GA7: gibe ellic acid; ABA: abscisic acid; and SA: salicylic acid

Figu e Click he e o access/download;Figu e;Fig1.jpg
Figu e Click he e o access/download;Figu e;Fig2.jpg
Figu e Click he e o access/download;Figu e;Fig3.jpg
Figu e Click he e o access/download;Figu e;Fig4.jpg