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Soil Biology & Biochemistry 78 (2014) 170e181

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Soil Biology & Biochemistry

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Soil organic matter degradation in an agricultural chronosequenceunder different tillage regimes evaluated by organic matter pools,enzymatic activities and CPMAS 13C NMR

M. Panettieri a, *, H. Knicker a, J.M. Murillo a, E. Madej�on a, P.G. Hatcher b

a Instituto de Recursos Naturales y Agrobiología de Sevilla, IRNAS-CSIC, PO Box 1052, 41080, Sevilla, Spainb Department of Chemistry and Biochemistry, Physical Sciences Building, Old Dominion University, 4402, Elkhorn Avenue, Norfolk, VA, 23529, United States

a r t i c l e i n f o

Article history:Received 23 February 2014Received in revised form27 July 2014Accepted 28 July 2014Available online 10 August 2014

Keywords:Conservation agricultureHumic acidsParticulate organic matterTotal organic carbon

* Corresponding author. Tel./fax: þ34 954624711.E-mail addresses: marco.panettieri@csic.es,

(M. Panettieri).

http://dx.doi.org/10.1016/j.soilbio.2014.07.0210038-0717/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Conservation agriculture (CA) is an important strategy to improve the quality of surface soils underMediterranean conditions and its primary intent is to minimize the mineralization of soil organic matter(SOM). The focus of this manuscript is to evaluate how soil quality indices (e.g., enzymatic activity, totalorganic carbon (TOC), and labile carbon pools) are inter-connected and how tillage could affect cycles ofstorage and degradation of SOM. To achieve this objective, five different enzymatic activities, TOC, watersoluble carbon and microbial biomass carbon were measured on soil samples collected at an experi-mental farm situated in the southwest region of Spain. Furthermore, the relative abundance of organiccompound classes was measured, using CPMAS 13C NMR on bulk soil samples, humic acids (HA), andparticulate organic matter (POM) fractions separated by density. Our results show that conservationtillage enhances soil quality at the surface for mid- and long-term experiments. The NMR analysesdemonstrate that conservation tillage led to a preservation of less degraded SOM and revealed a highamount of proteinaceous material preserved from degradation. It is clear that TOC, b-glucosidase andalkaline phosphatase activities are reliable soil quality indices and we further propose that the CPMAS13C NMR analyses of the particulate organic matter (POM) fraction of soil is an excellent evaluator forchanges that occur in soil status.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The use of deep plowing tools (e.g., moldboard) that lead to acomplete overturn of the soil is commonly defined as “traditionaltillage (TT)”. This type of tillage includes a sum of practices thatinvolves the removal of crop residues and their burial (or burning).Such a strategy has been widely blamed for erosion, soil degrada-tion, and organic carbon losses (Lal, 2005). The environmentalbenefits of implementing a different strategy of sustainable agri-culture are, among others, the maintenance and even theimprovement of the soil quality, the reduction of greenhouse gases,and the optimization of crop yields. Several reports have suggestedthe implementation of conservation tillage (CT), a strategy thatmaintains an ecological balance between economic and environ-mental targets and leads to a global improvement of soil quality at

marcopanettieri@gmail.com

the surface of soils (Moreno et al., 1997; Franzluebbers, 2004; Lalet al., 2007). Furthermore, CT promotes soil aggregation and pre-serves soil organic matter (SOM) from degradation (Six et al., 1999;�Alvaro-Fuentes et al., 2009; Panettieri et al., 2013). Such tillage doesnot involve the use of moldboards and reduces the number oftillage operations. In addition, for a correct establishment of CT, atleast 30% of the soil surface must be covered by crop residues afterharvesting (Gajri et al., 2002).

To assess soil conditions under different tillage systems, severalindicators of physical, chemical, or biochemical properties of soilare used (Franzluebbers et al., 1995; Madej�on et al., 2007). SOM andtotal organic carbon (TOC) contents are among those, and theirdistributions in the soil profile are the most common indices usedin tillage comparison experiments (Franzluebbers, 2002; Halvorsonet al., 2002). However, these measurements are occasionally notsensitive to changes that occur in soil, mainly those that are short-term (Rold�an et al., 2005). Furthermore, the sole use of SOM or TOCconcentrations provides little information on the degradation sta-tus of the SOM including soil carbon metabolism. To overcome this,specific operationally-defined fractions of SOM have been used as

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181 171

soil quality indicators. These include water soluble carbon (WSC)and microbial biomass carbon (MBC, �Alvaro-Fuentes et al., 2008;Melero et al., 2009), and these measurements determine theamount of readily bioavailable organic matter and the microbialcommunity activity, respectively. In contrast to TOCmeasurements,WSC and MBC measurements depend greatly on meteorologicalconditions, crop types, and methodology, situations that can limittheir reliability (Melero et al., 2011).

Enzymatic activity represents the most used early-responsefactor to evaluate soil quality (Nannipieri, 1994; Melero et al.,2008). Several aspects of carbon, nitrogen and nutrient cycles areoften evaluated by enzymatic essays. Some of these have been re-ported as trustworthy indices of soil status, and several studieshave demonstrated a strong seasonal and crop-dependence vari-ability (Feng et al., 2003).

Solid-state 13C NMR spectroscopy, using CPMAS, has been pro-posed by several authors as a valuable tool to assess the degrada-tion status of SOM (Golchin et al., 1994a; Baldock and Preston,1995;Preston, 1996; Knicker et al., 2012), because it allows the charac-terization of SOM composition noninvasively and without the useof solvents. However, the heterogeneity and complexity of SOMleads to broad resonance lines with overlapping peaks. The mainobjective of CPMAS 13C NMR spectroscopy is to obtain informationabout the relative contribution of different carbon functionalgroups to the total SOM of the sample.

In order to obtain reliable composite samples, it is important tolimit the spatial variability throughout the field at the sampling site(Knicker et al., 2012). Since interaction between paramagneticcompounds and SOM can affect the detectability of the carbon, theparamagnetics are often removed by demineralization with hy-drofluoric acid (Schmidt et al., 1997; Gonçalves et al., 2003; Knicker,2011). Because this also enriches the organic C, an increase of thesensitivity of the NMR experiment is realized. Other approaches forconcentrating the organic matter in the samples include theisolation of humic acids (HAc) and the extraction of particulateorganic matter samples (POM) (K€olbl and K€ogel-Knabner, 2004).However, this approach is not entirely a noninvasive one and weneed to consider these fractions of SOM as being only representa-tive of an operationally-defined part of the total material. Humicacids are defined as the fraction of SOM which is soluble in dilutealkali. Recent studies have demonstrated that the lower turnover ofthe HAc is not due to an intrinsic recalcitrance of these molecules,but it is more related to (i) their heterogeneity in size and chemistry(ii) associationwith soil mineral matter and (iii) physical protectiondue to the occlusion of SOM in aggregates that impedes microbialdegradation (Marschner et al., 2008; Kleber et al., 2011).

The POM fraction is separated by density fractionation and iscommonly described as a highly reactive portion of SOM. Thefraction not associated with minerals (f-POM) is mostly composedof fresh plant residues that act as easily degraded high-energyorganic materials (mostly carbohydrates and amino acids),whereas the occluded part of POM (o-POM) is strictly bonded to theclay fraction of the soil and represents a more aged and stabilizedfraction of SOM (Golchin et al., 1994b; Jastrow, 1996; John et al.,2005).

The aim of this work was to evaluate changes produced by 3different tillage strategies on the SOM in soils of a dryland exper-imental farm located in southwestern Spain with the intention toextract the most reliable soil parameters usable as indicators forsoil quality changes under our conditions. Different classical soilquality indicators and CPMAS 13C NMR spectroscopy are used as ameans to characterize the composition and degradation state of theSOM. The novelty of this experiment is in the evaluation of achronosequence of 3 different tillage comparison experimentsstarted at 3 different times in the same experimental area.With this

approach we intend to assess the response of chemical andbiochemical parameters to tillage practices and how tillage caninfluence the cycles of storage/degradation of SOM at differenttimes after its establishment. The soil of the experimental area is aCalcic Fluvisol (IUSS Working Group WRB, 2007). Three commonlyused chemical and biochemical parameters (TOC, WSC, and MBC)and five enzymatic activities were evaluated. A CPMAS 13C NMRanalysis was performed on bulk soil samples (BS), HAc, f-POM ando-POM.

2. Materials and methods

2.1. Experimental area and crop rotation

The present experiment was carried out at the “La Hampa”dryland experimental farm of the “Instituto de Recursos Naturales yAgrobiología de Sevilla (IRNAS-CSIC)” (37�170N, 6�30W), located13 km southwest of the city of Seville (Spain). The soil is a CalcicFluvisol (IUSS Working Group WRB, 2007) with a sandy clay loamtexture (240 g kg�1 clay, 180 g kg�1 silt and 580 g kg�1 sand). At adepth of 0e25 cm the soil has a high pH(H2O) of 7.8, an Olsenphosphorus content of 18.8 mg kg�1, a content of alkaline-earthcarbonates of about 280 g kg�1, and the organic carbon content isapproximately 9 g kg�1.

The climatic conditions are typically Mediterranean with mildrainy winters (496 mm mean annual rainfall) and very hot and drysummers. The mean annual daily temperature at the experimentalsite is around 19 �C, with maximum and minimum mean monthlytemperatures of 33.5 �C and 5.2 �C registered in July and January,respectively.

Since 1991, a wheat (Triticum aestivum L.) e sunflower (Heli-anthus annuus L.) crop rotation has been established. Subsequently,in 2005, a fodder pea crop (Pisum sativum L.) was included in therotation. Wheat receives deep fertilization with 400 kg ha�1 of acomplex fertilizer (15Ne15P2O5e15K2O) before sowing and a topdressing with 200 kg ha�1 urea (46% N), whereas sunflower andfodder pea crops are not fertilized. Since 2002, fertilization hasbeen reduced to 100 kg ha�1 (fertilizer complex) with no topdressing fertilizer. Weeds are controlled by tillage or by the appli-cation of pre-emergence herbicides depending on the tillagetreatment. A rate of 2 L ha�1 trifluraline (18%) was applied to thesunflower crop and 4 L ha�1glyphosate (18%) was applied to thewheat and fodder pea crop.

2.2. Tillage comparison experiments

An area of about 6000 m2 of this farm is designated for threedifferent experiments of tillage comparisons. Since 1991, an area ofabout 2500 m2 has been divided into 6 plots of approximately300 m2 (22 m � 14 m) each in a completely randomized experi-mental design (3 replicates per treatment). For this current exper-iment, traditional tillage (TTL) and reduced tillage (RTL) werecompared. Hereafter, the subscript (L) is used for long-termexperiments.

During the autumn of 2004, a different area of about 1500 m2

was divided into 6 plots of approximately 200 m2 (20 m � 9 m)each following the same completely randomized experimentaldesign (3 replicates per treatment) similar to the long-termexperiment. In these plots, a no-tillage management (NTM) iscompared to a traditional tillage (TTM). Hereafter, the notation mid-term experiment (M) will be used for this trial.

Finally, during autumn 2008, an additional area of about2000 m2 was divided into 9 plots of approximately 200 m2

(20 m � 9 m) each in a completely randomized experimentaldesign (3 replicates per treatment). For this trial, 3 tillage

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181172

treatments were compared: traditional tillage (TTS), reduced tillage(RTS) and no-tillage (NTS). Hereafter, the notation short-termexperiment (S) will be used for this experiment.

2.3. Tillage descriptions

The TT for each experiment consisted of a deep (25e30 cm)moldboard plowing with soil turnover (topsoil/subsoil inversion)followed by two cultivator passes to 15e20 cm depth and a discharrowing to 15 cm depth. Prior to 2003, crop residues wereburned, after which it was forbidden by the local government.Residues continued to be burned in 2004 and 2005 in the TT plotsthrough special permissions. TTS plots did not experience anyburning events, but crop residues were buried into the soil wherethe moldboard plow was used.

Within the RT plots for both long and short-term experiments,the number and depth of tillage operations was appropriatelyreduced, maintaining only chiseling to a depth of 15e20 cm, everyother year and a yearly disc harrowing to 5e7 cm depth, with largeamounts of crop residues left on the soil surface. The NT plot sur-faces were also covered by previous crop residues (except forsunflower stalks that are broken up into small pieces just beforesowing), no tillage operations are carried out and sowing is per-formed by direct insertion. The percentage of soil surface coveredby residue in both the RT and NT treatments was determined usingthe method described by Plaster (1992) and it was greater than 60%for all the crops used in rotations.

2.4. Sampling

For all experiments, soil samples were taken in early January2011, just before fodder pea was sowed. For each plot, one com-posite sample (composed of 5 different sampling locations withinthe same soil area) was collected for the depths: 0e5 cm, 5e10 cmand 10e25 cm. In total 63 samples were collected. The moist soilwas sieved through a 2 mm sized screen and divided into twosubsamples. One of thesewas immediately stored at 4 �C in a plasticbag loosely tied to ensure sufficient aeration and to preventmoisture loss before conducting enzymatic assays. The other sub-sample was air dried prior to chemical analyses that included solid-state 13C NMR.

2.5. Chemical analyses

Total organic carbon (TOC) of air-dried samples was measuredby dichromate oxidation and titration with ferrous ammoniumsulfate (Walkley and Black, 1934). Water soluble carbon (WSC)content was determined in a 1:10 (v/v soil:water) aqueous extractby horizontally shaking at 150 rpm for 1 h. The aqueous extract wascentrifuged using a fixed angle rotor in 40 mL tubes at 15 k rpm for10 min and measured using a TOC V-CSH Shimadzu analyzer.

2.6. Enzymatic activities

In the moist subsamples, microbial biomass carbon (MBC) and 5different enzymatic activities related to cycles of SOM and organicP, N, and S were evaluated. The content of MBC was determined bythe chloroform fumigationeextraction method modified byGregorich et al. (1990). Concentration of C in the extract wasmeasured by a TOC-VE Shimadzu analyzer and an extraction effi-ciency coefficient of 0.38 was used to convert the difference insoluble C between the fumigated and the unfumigated soil to MBC(Vance et al., 1987).

Dehydrogenases are a class of intracellular enzymes that catalyzetheoxidativedegradationprocess of theSOM, transferringhydrogen

and electrons from the organic substrates to acceptors. Dehydroge-nase activity (DHA) in soil was determined according to themethodof Trevors (1984) using the reduction of 2-p-iodo-nitrophenyl-phenyltetrazolium chloride (INT) to iodo-nitrophenyl formazan(INTF)measuring the absorbance at 490 nm to determine the extentof reduction. b-glucosidases are extracellular enzymes that catalyzethe hydrolysis of b-D-glucopyranosides releasing low molecularweight sugars. Alkaline phosphatase is an extracellular phosphoricmonoester hydrolase that regenerate inorganic P from organicsubstrates; similarly arylsulphatases catalyze the hydrolysis oforganic sulfate esters increasing the availability of inorganic sulfate.b-glucosidase activity (b-Glu), alkaline phosphatase activity (APA)and arylsulphatase activity (ASA) were determined with similarmechanisms, as described by Eivazi and Tabatabai (1988), Tabatabaiand Bremner (1969, 1970), respectively. Briefly, soil was incubatedfor 1 h with the corresponding substrate: (i) p-nitrophenyl-b-D-glucopyranoside for b-Glu, (ii) p-nitrophenyl phosphate disodiumfor APA, and (iii) p-nitrophenylsulphate for ASA. The p-nitrophenolproduced by the hydrolysis of these substrates was measuredspectrophotometricallyat 400nmabsorbance.Proteaseenzymesareresponsible of theproteolytic reactions andplayan important role inthe turnover of N in soil. Protease activity (PRA) was measured afterincubationof soilwith casein andmeasurementof the absorbanceofthe extracted tyrosine at 700 nm (Ladd and Butler, 1972). All thespectrophotometrical measurements were carried out on a PerkinElmer Lambda EZ210 spectrophotometer. Results for enzymatic andmicrobial parameters were based on the oven-dried weight of thesoil.

2.7. Isolation of SOM fractions

Humic acid extractionwas conducted by placing 20 g of soil in a250 mL centrifuge vessel and adding 60 mL of a 1 M NaOH solutionfree of CO2. The mixture was sonicated for 5 min, and subsequentlycentrifuged. The supernatant was collected and the extraction wasrepeated several times until the supernatant was no longer colored.The humic acid fractionwas isolated by lowering the pH to 1 addingHCl (10%) underN2flowand leaving the solution undisturbed for 8 hto allow the precipitate to settle. The precipitatewas separated fromthe solution by decanting after centrifugation (5 min, 3.5 k rpmusing a fixed angle rotor); these contained the humic acids whichare insoluble at low pH. The supernatant containing fulvic acids wasdiscarded. Afterwards the HAc isolates were placed into 10e12 kDacut-off tubular membranes and dialyzed against milliQ water untilthe conductivity remained constant and less than 50 mS cm�3

(Wander, 2004).The POM fractions were obtained by density fractionation of

bulk soil. In a wide plastic vessel, 50 g of soil were dispersed andslowly submerged beneath a 250 mL solution of sodium poly-tungstate (H2W12O40, MW: 2986,12 g mol�1) adjusted to a densityof 1.8 g cm�3. The mixture was gently shaken and allowed to settlefor 24 h. The floating f-POM was carefully collected, while theo-POM bound to clay was released by sonication (450 J mL�1) andseparated from the mineral phase by centrifugation (10 min 4 krpm using a fixed angle rotor). In order to eliminate polytungstateresidues, both f-POM and o-POM samples were washed withmilliQ water until the conductivity was below 5 mS cm�3 (K€olbland K€ogel-Knabner, 2004). All isolated BS, HAc and POM sam-ples were freeze-dried and ground to a fine powder beforeanalysis.

2.8. Solid-state 13C NMR spectroscopy

For each soil sample four different sets of subsamples wereprepared for NMR analyses: (i) bulk soil (BS), (ii) HAc, (iii) POM both

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181 173

as free and occluded fractions (f-POM and o-POM, respectively).Due to the low organic carbon content of deeper soil horizons, onlythe more organic-rich surficial samples (0e5 cm depth) wereanalyzed by NMR. For these analyses, air dried replicate sampleswere appropriatelymixed in order to obtain a composite sample foreach treatment, for a total of 7 samples.

In order to increase the NMR sensitivity of the bulk soil samples,they were demineralized to remove the mineral phase includingthe paramagnetics (Gonçalves et al., 2003). Briefly, 10 g of soil werefinely-ground, put into sealed polyethylene centrifugation vessels(250 mL) and treated four times with 40 ml HCl (10%) to eliminatemost of the calcium carbonate content and to avoid the formationof insoluble calcium fluoride during the subsequent HF deminer-alization process. After centrifugation and discarding the super-natant of the last HCl treatment, 40 mL 10% (v:v) HF solution wasadded. The mixture was shaken in the closed vessels for 2 h, andsubsequently centrifuged (10min 3 k rpm using a fixed angle rotor).The supernatant was removed and discarded appropriately. Afterrepeating the procedure four times, each sample was washed withdistilled water until the pH was above 5.

The samples were analyzed using an NMR spectrometeremploying the 13C CPMAS technique with a Bruker AVANCE II 400operating at a 13C frequency of 100.6 MHz. Approximately50e100 mg of sample was placed into a zirconium oxide rotor witha diameter of 4 mm and Kel-F caps. For all the measurements aspinning speed of 12 kHz was applied, the optimized contact timeand recycle delay were set to 1.5 ms and 1 s, respectively. About20 k scans were accumulated and a ramped 1H pulse was usedduring HartmanneHahn contact to circumvent HartmanneHahnmismatches.

For high quality spectra, Baldock and Smernik (2002) deter-mined that a 2% limit of significance for relative areas between thesame resonance ranges of two different samples should be takeninto account. However, Diekow et al. (2005) calculated differentlimits of significance for areas of each spectral region; differencesabove 8.3% in the relative area of carbonyl C, 5.0% for aromatic C,2.2% for O-alkyl C and 4.9% for alkyl C were considered significantfor their study. Other NMR studies performed by Knicker et al.(2012) on Brazilian soils demonstrated absolute deviations be-tween 1 and 25% for the different chemical shift regions of spectra

Table 1Values of relative intensity distributions (%) and standard deviations obtained from the

Time of establishment Treatment Carbox

Long-term RTL Sample 9.4Replicate 11.3SD 1.32

TTL Sample 10.4Replicate 12.2SD 1.25

Mid-term NTM Sample 8.6Replicate 11.2SD 1.82

TTM Sample 8.5Replicate 10.1SD 1.12

Short-term NTS Sample 12.4Replicate 12.2SD 0.12

RTS Sample 10.2Replicate 9.9SD 0.18

TTS Sample 12.4Replicate 13.0SD 0.41

of samples derived from different sites with the same soil type andcomparable soil management. Therefore, in order to account forpossible variations of the organic matter composition throughoutthe field, a composite sample was prepared for each treatment.

In order to elucidate the reliability of our NMR data, a second setof bulk soil samples collected in the same sampling period wasanalyzed with a Bruker AVANCE II 600 MHz spectrometer, oper-ating at a 13C frequency of 150.9 MHz. The spectra obtained withthis instrument were acquired with a spinning speed of 15 kHz, acontact time of 1 ms, a pulse delay of 400 ms, and the same numberof scans (20 k).

The samples of the second set were considered as replicates ofbulk soil samples and standard deviations of the obtained resultsare summarized in Table 1. Since differences between the twodiffered spectrometers were comparably low, no replicates wereanalyzed for HAc and POM samples. The obtained values are in therange reported within other studies (Baldock and Smernik, 2002;Diekow et al., 2005).

Spectra were evaluated with MestReNova version 8 (MestrelabResearch, Santiago de Compostela, Spain). All the FIDs weretransformed by applying a zero filling and an exponential filterfunctionwith a line broadening between 50 and 100 Hz dependingupon the sample being analyzed. A multipoint baseline correctionwas used. Quantification was performed by dividing the spectrainto 5 different chemical shift regions comprising 0e45 ppm,45e60 ppm, 60e110 ppm, 110e160 ppm and 160e220 ppm,assigned to alkyl C, methoxyl/N-alkyl C, O-alkyl C, aromatic C andcarbonyl/amide C, respectively.

2.9. Statistics

Data normality was tested by examining the ShapiroeWilk test.Significant differences between treatments due to tillage in thelong andmid-term experiments (RTL vs. TTL, and NTM vs. TTM) wereevaluated by a Student's t-test at p < 0.05. Soil differences for theshort-term experiments were analyzed by running a univariateanalysis of variance (ANOVA) to test whether therewas a significanteffect of the treatments (tillage) on each one of the studied vari-ables. Statistical analyses were carried out using IBM SPSS Statistics19.0 (SPSS Inc., Chicago, IL).

duplicates of bulk soil samples.

yl Aromatic O-alkyl N-alkyl Alkyl

21.4 29.2 9.5 30.518.6 28.4 11.8 29.81.93 0.53 1.66 0.50

24.7 29.7 9.6 25.622.4 29.9 10.3 25.21.60 0.13 0.52 0.29

17.9 33.3 11.5 28.714.7 31.9 12.9 30.22.26 1.04 1.00 1.05

20.1 29.0 10.9 31.418.4 28.9 12.4 30.01.25 0.05 1.12 0.98

18.8 30.4 10.8 26.416.5 30.4 12.8 28.11.66 0.01 1.39 1.24

18.4 30.0 11.2 29.015.6 30.3 12.8 31.41.97 0.22 1.10 1.67

19.5 29.2 9.6 29.416.0 29.2 12.3 29.52.45 0.01 1.96 0.08

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181174

3. Results

3.1. Chemical analyses

The conservation tillages increased the surficial TOC contents ofsoils and steadily rose to became more significant with timefollowing the start of the experiment (Table 2). For RTL trials, after20 years of establishment, a significant increase of TOC of approx-imately 30% at the surface (0e5 cm) and at 5e10 cm was observedwhen compared with TTL trials (Table 2). In the mid-term experi-ments, NTM showed significantly higher TOC contents than TTM, butonly at the surface (0e5 cm) and with lower magnitude than in thelong-term comparison. No significant differences in TOC contentswere found for the short-term experiments, although the NTS trialwas the most effective treatment for TOC accumulation at thesurface, whereas TTs showed higher TOC values deeper in the soil.No significant differences in WSC were observed between thetreatments or experiments. Significant differences for MBC werefound only for RTL at 0e5 cm depth when compared with TTL. Forlong and mid-term experiments, MBC content was always higherbut not at a significant level in the conservation tillage treatments,whereas a clear trend was not recognizable for the short-term ex-periments (Table 2).

3.2. Enzymatic activities

All enzymatic activities showed significantly higher values forthe topsoils of RTL when compared with those of TTL except for PRAthat showed slightly but not significantly higher values than RTL.Moreover, significant differences for RTL were found also at5e10 cm depth for b-Glu and ASA. Deeper in the soils (10e25 cm),all the measured activity values under TTL were slightly but notsignificantly higher than for RTL (Table 2).

For the mid-term experiments, NTM surface soils showed sig-nificant differences for b-Glu, APA and ASA. At deeper horizons(5e10 and 10e25 cm) no significant differences were found,although higher activity levels were measured for NTM samples

Table 2Mean values of total organic carbon (TOC, g kg�1), water soluble carbon (WSC,mg kg�1), mh�1 DWsoil), b-glucosidase activity (b-glu, mg p-nitrophenol kg�1 h�1 DW soil), alkaline p(ASA, mg p-nitrophenol kg�1 h�1 DW soil), and protease activity (PRA, mg tyrosine kg�1 h�

at three depths (0e5, 5e10 and 10e25 cm) for the three experiments of long-, mid-, and sor different letters (p < 0.05).

Time of establishment Depth Treatment TOC WSC

Long term 0e5 cm RT 13.9* 112TT 10.8 107

5e10 cm RT 13.0* 119TT 10.4 100

10e25 cm RT 8.2 94TT 9.4 90

Mid term 0e5 cm NT 10.8* 118TT 8.9 103

5e10 cm NT 9.4 119TT 8.6 111

10e25 cm NT 6.7 90TT 8.1 82

Short term 0e5 cm NT 10.1 117RT 9.0 104TT 9.1 128

5e10 cm NT 8.5 103RT 8.2 102TT 9.0 119

10e25 cm NT 6.8 92RT 6.3 85TT 7.1 80

when compared with TTM. Exceptions were found for ASA and PRAat 10e25 cm (Table 2).

Significant differences for the short-term experiments werevaried. At the surface, NTS showed higher b-Glu values whencompared with TTS and showed higher APA when compared withRTS. The values of other enzyme activities were higher for NTS butnot significant compared with the other treatments (Table 2). TheRTS samples had significantly higher APA values at 5e10 cm depthwhen compared with NTS that had higher ASA values than TTS at10e25 cm depth.

Generally, a stratification trend can be identified for all thetreatments, in which activity levels are higher at surface anddiminished with depth, although this trend was less clear for plotsunder traditional tillage. Significant differences for the enzymaticactivities were mainly found at surface, where the activity levelswere higher (Table 2). APA and b-Glu were the two activities thatresponded the best to changes produced by tillage, since theyshowed significant differences at the surface for the three experi-ments. In the topsoil, ASA values showed no significant differencesfor the short term experiments; whereas DHA values were signif-icant only for long term experiments and PRA values were statis-tically insignificant for all cases (Table 2).

3.3. 13C nuclear magnetic resonance spectroscopy

The superposed 13C CPMASNMR spectra obtained for BS, HAc, o-POM and f-POM shown in Fig. 1 are for the samples of RTL. Com-parable spectrawere obtained for the other treatments. The relativeintensity distributions among the different chemical shift regionsof the spectra are presented as histograms (Figs. 2e4). The domi-nant signal in the spectra of BS and of both POM fractions, espe-cially for the f-POM, is identified in the region between 110 and60 ppm, which is assigned to O-alkyl C. High intensities corre-sponding to the alkyl C (45e0 ppm) and aromatic C (160e110 ppm)were detected for o-POM, HAc and BS samples. HAc, and to a minorextent, o-POM showed a clear phenolic C signal (160e140 ppm)that typically derives from lignin. A strong signal between 60 and

icrobial biomass carbon (MBC, mg kg�1), dehydrogenase activity (DHA,mg INTF kg�1

hosphatase activity (APA, mg p-nitrophenol kg�1 h�1 DWsoil), arylsuphatase activity1 DW soil) in soil under no tillage (NT), reduce tillage (RT) and traditional tillage (TT)hort-term. Significant differences between treatments are indicated with an asterisk

MBC DHA b-Glu APA ASA PRA

580* 1.25* 225* 362* 36.3* 129474 0.53 101 259 18.9 99627 0.89 132* 246 29.0* 53530 0.61 75 228 16.9 70588 0.32 41 216 18.8 39524 0.41 54 208 19.8 60

687 1.05 160* 306* 33.6* 126670 0.79 81 222 12.1 102719 0.66 137 246 29.0 68604 0.61 80 217 27.0 65644 0.44 29 209 23.4 47626 0.43 23 146 25.4 54

746 1.17 116 b 310 b 55.0 153958 1.04 87 ab 191 a 39.4 152753 0.71 63 a 260 ab 37.5 107667 0.38 a 38 a 183 a 46.3 105881 0.51 ab 55 ab 252 b 43.8 80868 0.69 b 76 b 244 ab 42.0 125581 0.44 30 ab 171 50.2 b 51740 0.41 14 a 154 38.8 ab 50689 0.44 45 b 185 35.3 a 62

Fig. 1. CPMAS 13C NMR stacked spectra of the topsoil (0e5 cm) collected from the RTL plot of “La Hampa” dryland experimental farm. Bulk: bulk soil demineralized with HF; HumicAc.: humic acid fraction; o-POM: particulate organic matter-occluded fraction; f-POM: particulate organic matter-free fraction. d: chemical shift; *Aromatic C region spinning sidebands; **O-Alkyl C region spinning side bands.

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181 175

45 ppm typically assigned to N-alkyl C or methoxyl C, was found forBS, o-POM and HAc.

Comparing the organic matter composition of the BS samples inrelation to the soil management, higher aromatic C concentrationswere observed for the traditional tillage of long- and mid-term ex-periments when compared with the corresponding conservationtreatments, even if differences were more obvious for the long-termexperiment. No major changes are apparent for the N-alkyl C andCarboxyl C contents of the long- and mid-term experiments. The in-tensity of the O-alkyl C region was higher for NTM when comparedwith thatof TTM,whereas RTL andTTL had the same intensities for thisregion. TT treatments showed a higher intensity of alkyls atmid-termanda lower intensity for the sameregion inthe long-termexperiment.For the short-termsamples, only fewdifferenceswere foundbetweentreatments. TTS had the lowestN-alkyl andO-alkyl intensities and thehighest content in aromatic,whereasNTS showed the highest contentof O-alkyls and the lowest intensity for the alkyl region.

For HAc samples from the long-term experiment, the spectra ofthose from RTL showed higher intensities for carboxyl and aromaticC regions when compared with those of TTL, whereas the latter hadhigher intensity for the O-alkyl C region. No differences were foundfor N-alkyl C regions. For the mid-term experiment, the spectra ofNTM showed a higher content of alkyl C and lower intensities foraromatic and carboxylic C, if compared with those of TTM. For O-alkyl C and N-alkyl C no differences were observed.

The short-term experiment confirmed the trends for alkyl C, N-alkyl C, and aromatic C for the extracts of the long- and mid-termexperiments, in which NT had higher contributions of alkyl C andRT had the highest aromatic C contents, whereas no differenceswere found for N-alkyl C. Despite the other experiments, NTS hadmore carboxylic C and RTS had more O-alkyl C at short-term ifcompared with corresponding TTS samples.

The spectra obtained for f-POM samples showed a clear pattern.Conservation tillages displayed the highest O-alkyl concentrations

Fig. 2. Relative intensity distributions of the various bands in 13C NMR spectra ob-tained from the bulk soil (0e5 cm) treated with HF of “La Hampa” dryland experi-mental farm. The three different times of establishment experiments are compared.NT: no tillage, RT: reduced tillage; TT: traditional tillage.

Fig. 3. Relative intensity distributions of the spectral bands in 13C NMR spectra ob-tained from the humic acids extracted from the topsoil (0e5 cm) of “La Hampa”dryland experimental farm. The three different times of establishment experiments arecompared. NT: no tillage, RT: reduced tillage; TT: traditional tillage.

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181176

at mid- and long-term if compared with TT, whereas at short-term,differences became less noticeable for this carbon functional group.Alkyl C contents showed an inverse pattern with time, comparedwith O-alkyl carbon, since values obtained were higher for TT atshort- and mid-term but not at long-term, were RTL had highervalues than TTL. Aromatic and carboxylic contributions weregreater for TT at long- and mid-term but not at short-term, whereTTS had the lowest values if compared to NTS and RTS. Differencesfound for N-alkylic C were less evident, except for o-POM at short-term, where NTS showed 2% lower intensities than TTS.

4. Discussion

Several studies have reported that enzymatic activities areearly-response factors and useful indicators for the evaluation ofanthropogenic and natural changes occurring in soil (Nannipieri,1994; Benedetti and Dilly, 2006; Melero et al., 2008). No studies

were performed to evaluate how the reliability of enzymatic andchemical parameters varies in an agricultural chronosequencefarm under different tillages. The results of this study indicatethat enzymatic activity response to changes in soil characteristicsis more reliable at the soil surface, where the micro-environmentis richer in oxygen and contains a higher amount of organicmatter derived from the degradation of plant residues. Enzymeproduction is influenced by numerous factors, and the evaluationof this parameter always depends on the soil characteristics,microbial populations, plant residues, roots exudates, and micro-or macro-climatic conditions (Feng et al., 2003; Melero et al.,2011).

In our study, only some of the evaluated activities showed sig-nificant differences at the surface for all three experimental times.The long-term experiment, showed the biggest differences be-tween the two tillages under evaluation; RTL had significantlyhigher values for enzymatic activities but also for MBC and for TOCat surface.

Fig. 4. Relative intensity distributions of the 13C NMR spectra obtained from the particulate organic matter fractions separated from the topsoil (0e5 cm) of “La Hampa” drylandexperimental farm. The three different times of establishment experiments are compared. NT: no tillage, RT: reduced tillage; TT: traditional tillage; f-POM: particulate organicmatter-free fraction; o-POM: particulate organic matter-occluded fraction.

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181 177

This study indicates that the use of conservation tillage im-proves TOC accumulation in the topsoil and promotes a stratifica-tion of organic carbon that could greatly enhance soil quality, asreported also by Franzluebbers (2002) and Mrabet (2002). Conse-quently, treatments with higher organic carbon contents in thetopsoil were also characterized by higher levels of microbial ac-tivity, in agreement with other studies (Six et al., 2000; �Alvaro-

Fuentes et al., 2008). Subsoil samples from the same experi-mental area showed a partial redistribution of TOC under TT, butconfirmed a net carbon accumulation under conservation treat-ments (L�opez-Garrido et al., 2011).

TOC contents showed significant differences only for long- andmid-term experiments, since this parameter gives a significantresponse only after prolonged experimental times (Rold�an et al.,

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181178

2005). Furthermore, with increasing time after the establishmentof the experiment, some parameters (TOC, b-Glu and ASA) showedsignificant differences also at a depth of 5e10 cm. This is most likelydue to the partial redistribution of surface soil induced by chiselplowing in the RTL plots. Differences with respect to b-glu weresignificant for the topsoil both in NTM and in NTS if compared withTTM and TTS, which suggest that b-glu is a reliable indicator of soilquality. Similar findings were reported by other studies in the samegeographic area (Madej�on et al., 2007; Melero et al., 2011;Panettieri et al., 2013). The higher amount of plant residues left atsurface under conservation treatments provides a higher quantityof polysaccharide substrate that results in a higher b-glu activity.

For the mid-term experiment, TOC values were higher for NTMsamples than for the corresponding TTM samples. No-tillage pro-voked an enhancement of TOC at the surface even at mid-term, dueto the high quantity of residues that cover the plots and the absenceof tillage. These factors strongly diminish the degradation of SOM,presumably by forming large aggregates that protect SOM frommicrobial and chemical oxidation. This effect is also observed inother types of CT, but soils under NT have the highest tendency toform macroaggregates (Six et al., 1999; Plaza-Bonilla et al., 2010).

Activities such as APA and ASA maintained their levels at mid-term, and may also be considered as viable soil quality indicatorssince both activities were described as being strongly related toSOM augmentations (Deng and Tabatabai, 1997). The significantdifferences in APA values for the short term experiment, in whichNTS had significantly higher values than RTS, demonstrated that thisparameter reflects changes in SOM more rapidly than TOC. Incontrast, ASA did not show significant differences at short times,due to the high variability of the data.

The significantly higher values of DHA and b-glu found at deepersoil horizons for TTS samples compared with NTS may be explainedby soil inversion produced by the moldboard approach to tillage.NTS maintained constant ASA values whereas other treatmentsshowed a diminution of values with depth, inducing a significantlyhigher value of ASA for NTS than for TTS at 10e25 cm depth.

As reported byMelero et al. (2011) MBC can vary strongly due toexternal conditions, and DHA appears to be too readily influencedby meteorological fluctuations for being considered as a viable soilindicator in our study. Significant differences for these parameterswere found only for the long term experiment.

Values measured for PRA activity can be related to the strongsignal at 53 ppm of the CPMAS 13C NMR spectra especially for HAcsamples, but also for BS, in all treatments. This signal is partlyderived from N-alkyl C, most tentatively peptides. FT-IR spectros-copy (data not shown) confirms this assignment by a wide ab-sorption band at 3700e3500 cm�1, typically assigned to the amideNeH stretching vibration (amide A band) as well as the othertypical peptide bands (amide I and II) at approximately 1650 and1540 cm�1, respectively (Krimm and Bandekar,1986). Since NaOH iscommonly used to extract proteins from soil samples (Benndorfet al., 2007), the HAc isolation process could have led to anenrichment of peptides in HAc samples. The high intensity of sig-nals assignable to peptides in the spectra of the bulk soil could berelated to the presence of encapsulated or protected peptides thataremore recalcitrant to degradation. The signal at 53 ppm is usuallyderived from methoxyl C of lignin (Hatcher, 1987) but may also beassigned to the alpha carbon of peptides. However, the relativelylow intensity between 140 and 160 ppm indicates that lignincontributions are not large and confirms that the main source ofsignal at 53 ppm is due to peptide C (Knicker and Hatcher, 1997).Possibly, such peptide material could have been protected byadsorption to minerals (o-POM) or in the humic acid network froma rapid degradation due to PRA activity; Miltner et al. (2012) pro-posed the entrapment in patchy fragments derived from cell walls

as a possible protection mechanism of cytoplasmic material. ThePRA values found for this experiment were similar to those foundfor other studies (Ladd and Butler, 1972; Trasar-Cepeda et al., 2000;Marinari et al., 2006).

Chemical and biochemical analyses demonstrated that,compared to soils under TT, the implementation of CT progressivelyimproved the quality of superficial soil with increasing time. Thestorage and the preservation of SOM in CT, together with a differentlevel of enzymatic activities could lead to a selective enrichment ofdifferent classes of organic compounds in soils from the same areaunder different tillages.

The results shown in this study indicated that the compositionof bulk soil is more comparable to the NMR pattern found for HActhan to the one observed for POM samples. The 5 spectral regionscontain a certain number of peaks that could be normally assignedto the main class of compounds present in each region (Fig. 1). Thesignals in the O-alkyl C region (110e60 ppm) are typically assignedto carbohydrates derived mostly from cellulose and bacterialbiomass, with a minor contribution of ethers and proteins (Prestonand Trofymow, 2000). This material is commonly easily metabo-lized by soil organisms. The fact that the O-alkyl C region dominatesthe spectra of POM samples (Fig. 4) confirms that f-POM is definedas SOM at an early state of decomposition (Golchin et al., 1994a;John et al., 2005).

The aromatic C region embraces intensities of C in lignin resi-dues, aromatic amino acids and black carbon. For the BS samples ofour study, the spectra indicate an increase in aromaticity withincreasing experiment duration, and particularly in TT samples(Fig. 2). This could be related to two different factors that occur to adifferent extent for RTL and TTL: (i) the continuous agriculturalpractices of RT leads to fast degradation of fresh material rich in O-alkyl C leading to a relative enrichment of lignin residues and (ii)the accumulation of pyrogenic organic matter, since burning of thecrop residues following the harvest was carried out until 2005 onthe TT plots of the long-term experiment and to a smaller extentalso on the plots of mid-term experiments (Gonz�alez-P�erez et al.,2004; Knicker, 2007). The short term experiment did not experi-ence any recent burning event and values of aromatic contents arecomparable between all the 3 treatments.

A lower O-alkyl C content was revealed for the BS samples of theNTM compared with those of TTM, indicating that the SOM of thelatter was more degraded. The relative contribution of alkyl Ccompounds tends to increase in highly exploited soils with lowinput of litter rich in cellulose (K€ogel-Knabner and Ziegler, 1993;Baldock and Preston, 1995). However, for the long term experi-ment, the alkyl C content was lower for TTL than for RTL, most likelybecause the input of aromatic structures with the burned residueswas higher than the relative preservation of alkyl C in TTL samples.

The alkyl C to O-alkyl C ratio was suggested as an index for thedegradation or oxidation status of SOM (Baldock and Preston,1995). However, this index is viable only for comparisons be-tween samples from analogous areas and with comparable SOMsources. For the samples of the studied experimental area, the alkylC to O-alkyl C ratio ranged between 0.84 and 1.08 (Table 3). NTM,NTS, and TTL samples showed the lowest ratios confirming thatmore fresh litter is entering and accumulating in the soils of the NTtreatments under similar conditions (Panettieri et al., 2013). A se-lective enrichment of black C compounds that are less easilymetabolized by microorganisms and that connect intimately withthe clay fraction of mineral soil was found for TTL plots (Marschneret al., 2008). Concomitantly, TTL showed a lower content of alkyl Cthan the same treatment at short and mid-term. This could berelated to the effect of residue burning and to the lower MBCcontent found in the previous years for TTL if compared to RTL(Murillo et al., 2004; Madej�on et al., 2007; L�opez-Garrido et al.,

Table 3Alkyl C to O-alkyl C ratios obtained for the different samples.

Time of establishment Treatment Alkyl C/O-alkyl C

Long-term RTL 1.05TTL 0.86

Mid-term NTM 0.86TTM 1.08

Short-term NTS 0.87RTS 0.97TTS 1.01

M. Panettieri et al. / Soil Biology & Biochemistry 78 (2014) 170e181 179

2009; Madej�on et al., 2009). Plaza et al. (2013) have recentlypointed out the importance of soil biomass and necromass for long-term C storage, and a lower amount of “patchy” fragments rich inlipid membranes derived from cell walls could have resulted in alower alkyl C content (Miltner et al., 2012).

The better physical quality of soils under NT and the higher SOMquantity resulted in an overall improvement of the soil quality andin a better preservation of SOM, as demonstrated by enzymaticactivities related with SOM degradation, such as b-glu and APA, bythe higher TOC contents found in NT samples, and by the lessdegraded SOM present in NT plots.

Humic acids from RTL and RTS were richer in carboxyl and ar-omatic C than corresponding TT plots, probably due to the higheramount of partially decomposed plant residue that could embed ahigher quantity of degraded lignin into the humic acids (VonLützow et al., 2008). Such a pattern was not observed for aro-matic C of the NTM and NTS plots against TT plots, since the absenceof tillage could have limited the interaction of fresh plant residueswith humic substances and clays. The higher abundance in alkyl Cfor NTM and NTS than for TT plots is mainly due to the higher mi-crobial biomass content that could have enriched the humic acidsfraction with necromass residues and cell wall fragments (Miltneret al., 2012). Analyzing the evolution in time of the HAc from TTplots, a depletion of alkyl C was observed, followed by an enrich-ment of carboxyl C at mid-term and O-alkyl C at long term.Furthermore, amide C may also contribute to the carboxyl C region(Skjemstad et al., 1983). This could suggest a possible microbialoxidation pattern of alkanes in absence of a more readily metabo-lizable substrate.

The higher amount of residues accumulating at the soil surfaceduring conservation treatments confers fresh SOM substrate to thesoil; this was reflected by the higher abundance of O-alkyl C in f-POM samples from RTL and NTM when compared with TTL and TTM,respectively. However, differences are less evident for the short-term experiment, probably due to the shorter time of establish-ment. TTS had a greater abundance of aromatic constituents atlong- and mid-term, and higher abundance of alkyl C at short-andmid-term when compared with the corresponding NTS and RTStreatments.

As expected, O-alkyl C contents of o-POM samples decreased,since o-POM is a more degraded SOM fraction strongly connectedto the clay fraction of the soil (Golchin et al., 1994a). In contrast,alkyl and aromatic C regions increased their relative abundances ino-POM compared with f-POM as confirmed by other studies (Plazaet al., 2012). The spectra of the o-POM samples of RTL and NTM hadhigher intensities in the O-alkyl C region when compared with thespectra of the corresponding TT treatments, similar to what wasobserved for the f-POM. Even for the short-term experiment, NTSand RTS followed the same pattern if compared with TTS. Theopposite tendency was noted for aromatic C, which showed highercontributions for TT samples after longer times of establishment.The aromatic compounds may have derived from the residues

associated with burning practices continued in the long- and mid-term experiment, and as stated previously, the evidence forburning events is confirmed by NMR spectra. Following the chro-nosequence for both POM fractions, it seems to rapidly respond tochanges in soil management, especially its O-alkyl C content, sug-gesting the chemical composition of this fraction as a viable indi-cator of soil quality.

5. Conclusion

The results obtained suggest the use of enzymatic activities suchas b-glu, APA and to a minor extent ASA, as early indicators of soilquality at surface, whereas TOC and MBC could be viable for long-or mid-term comparisons. Results obtained strongly suggest theadoption of conservation tillages to improve soil chemical andbiochemical properties at the soil surface and to enhance the inputsof fresh SOM.

CPMAS 13C NMR revealed an accumulation of aromatic struc-tures in plots experiencing residue burning, and a high amount ofproteinaceous material in BS, HAc, and o-POM fractions. Furtherresearch is needed to understand how these structures are inter-actingwith each other andwith themineral phase of soil in order toexplain their preservation from degradation.

Moreover, the results obtained for CPMAS 13C NMR suggesteduse of o-POM and f-POM as the most relevant soil fractions in orderto evaluate the degradation status of soil under different tillages.

Acknowledgments

The authors would like to thank the anonymous reviewers fortheir valuable comments and suggestions to improve the quality ofthe paper.

The authors would like to thank the Interministerial Commis-sion of Science and Technology (CICYT) through project AGL2010-22050-C03-03, which allowed this work to be carried out, and theDepartment of Chemistry and Biochemistry of the Old DominionUniversity (Norfolk, VA, USA) which hosted the senior authorduring his grant and provided access to the NMR facility of theCollege of Science Major Instrumentation Cluster (COSMIC).

Dr. Rosa L�opez-Garrido is acknowledged for her technicalsupport.

M. Panettieri thanks European Social fund (ESR), Consejo Su-perior de Investigaciones Científicas (CSIC), and the Ministry ofEconomy and Competitiveness of Spain for funding his Ph.D. grant(JAEPre_09_01448) and his stay at the Department of Chemistryand Biochemistry of the Old Dominion University (Norfolk, VA,USA).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.soilbio.2014.07.021.

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