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ISSN 0178-2762, Volume 46, Number 5

ORIGINAL PAPER

Reversing agriculture from intensive to sustainable improvessoil quality in a semiarid South Italian soil

Luigi Badalucco & Maria Rao & Claudio Colombo &

Giuseppe Palumbo & Vito Armando Laudicina &

Liliana Gianfreda

Received: 13 November 2009 /Revised: 17 February 2010 /Accepted: 22 February 2010 /Published online: 27 March 2010# Springer-Verlag 2010

Abstract Intensive agriculture (IA) is widespread in SouthItaly, although it requires frequent tillage, large amounts offertilizers and irrigation water. We have assessed theefficacy of reversing IA to sustainable agriculture (SA) inrecovering quality of a typical South Italy soil (LithicHaploxeralf). This reversion, lasting from 2000 to 2007,replaced 75% of nutrients formerly supplied inorganicallyby farmyard manuring and reduced the tillage frequency.Several chemical and biochemical properties, functionallyrelated to C and N mineralisation–immobilisation processesand to P and S nutrient cycles, were monitored annuallyfrom 2005 to 2007 in the spring. Reversing IA to SAdecreased soil bulk density, almost doubled the soil organicmatter (SOM) as favoured the immobilisation of C and N,increased most soil microbial indicators but decreasedcontents of nitrate, mineral N and K2SO4-extractable C.The K2SO4-extractable C/K2SO4-extractable organic Nratio suggested that substrate quality rather than the massof readily available C and N affected biomass and activity

of soil microflora. Also, the largely higher 10-day-evolvedCO2–C-to-inorganic N ratio under SA than IA indicatedthat higher C mineralisation, associated with highermicrobial biomass N immobilisation, occurred under SAthan IA. Decreases in most soil enzyme activities under IA,compared to SA, were much higher than concomitantdecreases in SOM content. Soil salinity and sodicity werealways higher in IA than SA soil, although not criticallyhigh, likely due to the intensive inorganic fertilisation asirrigation waters were qualitatively and quantitatively thesame between the two soils. Thus, we suggest that thecumulative small but long-term saline (osmotic) and sodic(dispersing) effects in IA soil decreased the microbialvariables more than total organic C and increased soil bulkdensity.

Keywords Soil quality . Sustainable agriculture . Soilmicrobial biomass .Microbial activity . SemiaridMediterranean environment

Introduction

Soil microbial properties are more sensitive than physicaland chemical properties in detecting changes in soil quality(Doran et al. 1996; Nannipieri et al. 2003). Agriculturalmanagement is one of the most significant anthropogenicactivities that greatly alter soil quality (van Diepeningen etal. 2006). Soil biota, a small, labile but crucial fraction ofsoil organic matter (SOM), is the driving force behindenergy transfers and nutrient transformations in soils, thusplaying a major role in soil fertility and resiliency (Elliott etal. 1996). Soil enzyme activities, as effectors of soilmicrobial activity, may represent reliable indicators of soilquality as they show a quick response to the changes

L. Badalucco (*) :V. A. LaudicinaDipartimento di Ingegneria e Tecnologie Agro-Forestali,Università di Palermo,Viale delle Scienze 13,90128 Palermo, Italye-mail: badalucc@unipa.it

M. Rao : L. GianfredaDipartimento di Scienze del Suolo, della Pianta, dell’Ambientee delle Produzioni Animali, Università di Napoli Federico II,Via Università 100, Portici,80055 Naples, Italy

C. Colombo :G. PalumboDipartimento di Scienze Animali, Vegetali e dell’Ambiente,Università del Molise,Via De Santis,86100 Campobasso, Italy

Biol Fertil Soils (2010) 46:481–489DOI 10.1007/s00374-010-0455-y

Author's personal copy

induced by both anthropogenic and natural disturbances(Dick 1992; Goyal et al. 1993; Garcia and Hernandez 1997;Izquierdo et al. 2005).

Maintaining SOM is a major task in modern agriculture,since farming practices such as high vegetable productionand frequent soil tillage may lead to its reduction. InMediterranean semiarid countries, this problem is evengreater as high mean temperatures promote high mineralisa-tion rates of organic matter and favour the degradation ofcropped soils through low rainfall and high evaporativedemand. Rural landscapes in South Italy are often charac-terised by intensive agriculture (IA), with use of largeamounts of fertilizers and irrigation water. Long-terminorganic fertilizer applications in intensive irrigated agricul-ture can have either deleterious or beneficial effects on soilmicrobial and biochemical activity (Biederbeck et al. 1996;Simek et al. 1999; Böhme et al. 2005; Deng et al. 2006).

Low-input sustainable agriculture, based on the reduc-tion of chemical inputs and practices to increase soilorganic matter levels, can be a suitable method of managingsoil quality, but in southern Italy, it is a hardly usedmanagement (Iovieno et al. 2009). The aim of this workwas to assess the efficacy of reversing intensive agriculture(high inorganic fertilisation and frequent tillage) to a moresustainable management, in order to recover quality of atypical soil (Lithic Haploxeralf) in southern Italy.

Materials and methods

Study site description, soil sampling and experimentaldesign

The study was carried out in a semiarid area of southernItaly (Puglia Region, Bari Province) at Ripagnola (41°3′ Nlatitude and 17°3′ E longitude). The soils, developing onhard limestone, are quite shallow (0–25 cm), heavy-textured and slow-draining, although never hydromorphic,and belong to Lithic Haploxeralfs (Soil Survey Staff 2006).The land surface is gently rolling and generally intensivelycultivated with vegetable crops.

The climate of the area is Mediterranean with warm drysummers and mild rainy winters. As evaporation usually farexceeds precipitation during the growing season, irrigationis necessary to maintain regular plant growth, especially inintensive conventional agriculture. At the study site, meanannual temperature is about 16°C, the warmest month isAugust (26.4°C) and the coldest January (7.4°C). Meanannual precipitation during the 3-year investigation periodwas about 530 mm, with a maximum in December (82 mm)and a minimum in July (9 mm). Consequently, soilmoisture and temperature regimes are xeric and thermic,respectively.

At the site, two contiguous square fields, both 1.4 hawide and kept fallow from 1986 to 1991, were intensivelycropped since 1992. The first field (IA) annually producedtomato or eggplant in spring and celery, lettuce, parsley,broccoli, cauliflower or spinach in summer–autumn, till2007. It was frequently tilled and fertilised with400 kg ha−1 year−1 of N P K (15%, 18%, 20%,respectively) and 50 kg ha−1 year−1 of S. Moreover, anumber of different conventional pesticides were appliedfor an effective control of fungi (sulphur 80%, copperoxychloride, mancozeb 40% and cymoxamil 4%), nemat-odes (metam sodium) and weeds (glyphosate). This cropmanagement schedule was based on decisions made by theregional grower and the pest control advisor, as recom-mended by the Italian Ministry of Agriculture for that area.The irrigation water was moderately saline and sodic(electrical conductivity (EC) from 1.2 to 2.5 dS m−1, SAR6–9), as was taken from an artesian well 10 m below sealevel, and was applied for 4–6 h twice a week by a dripsystem (0.8 l/h), so as to deliver 20–40 mm, depending onthe season, of water per hectare per week during eachvegetable growth cycle, i.e. about 200–400 m3 ha−1

week−1. At the IA field, the tillage was rather intensivesince the operations could include ripping at 20–40 cmdepth, disking, power harrowing, landplaning and bedshaping, depending on the cropped vegetables.

The second field (sustainable agriculture (SA)) wasmanaged like IA field between 1986 and 1999 but since2000 was converted to more sustainable agriculture, due toa significant reduction in vegetable yield. Thus, theagriculture management of the SA field differed from theIA field between 2000 and 2007, as inorganic fertilisationrate was lowered by 75%, and the missing amounts of N, P,K and S were supplied as farmyard manure (between 3 and4 t ha−1 year−1 depending on its composition), which wasderived from chicken manure and leguminous residues(Vicia faba and Vicia sativa), mixed approximately at thesame rate and left in the open air about 1 year. The dry bulkdensity of the used farmyard manure between 2000 and2007 was 0.34±0.08 kg dm−3, whereas total N and total Pcontents were 2.9±0.3% and 1.8±0.3% on a dry weightbasis, respectively. The farmyard manure was annuallyincorporated into soil to a depth of 20 cm in summer bydisk ploughing. Also, tillage frequency was reduced asmuch as possible (at least annually halved in comparisonwith IA field), especially when soil aggregation appearedvisually improved. Sustainable management (SA) differedfrom IA only for manuring and reduced tillage, as irrigationwaters and pesticide application rates were the same both inquantity and quality. Both IA and SA fields were sampledon 14 April 2005 (I), 20 April 2006 (II), and 24 April 2007(III) as follows: We selected five plots (each 3×3 m wide)placed at the five discontinuity points of a W-shaped

482 Biol Fertil Soils (2010) 46:481–489 Author's personal copy

transect wholly horizontally representing each field, accord-ing to a non-systematic sampling design. Four 3-kg sub-samples were collected from the 5–15 cm depth in each plotand pooled together to give a fresh composite soil sample.The 0–5 cm soil layer was discarded to eliminate the driersoil and reduce spatial variability. Therefore, 15 soilcomposite samples (5×3 annually repeated samplings)were obtained from each field in the 3 years. Soil sampleswere either freshly (biological properties) or air-driedsieved (physico-chemical properties) to <2 mm and storedat +4°C before analyses.

Soil physical and chemical analyses

Soil moisture content was determined gravimetrically (soildried overnight at 105°C) and used to express all data on adry weight basis. Actual and potential acidity weredetermined potentiometrically by a glass membrane elec-trode in 1:2.5 (w/v) soil/distilled water and 1 N KClmixtures, respectively. EC was measured in 1:2.5 (w/v)soil/distilled water extracts. Particle size distribution wasdetermined by the pipette method, and bulk density of field-moist soil cores was determined according to Soil SurveyLaboratory Staff (1996). Total organic C (TOC) wasdetermined by wet-acid dichromate oxidation and total Nby the Kjeldahl method. Exchangeable basic cations (EBC)were extracted for 2 h with 1 M BaCl2 buffered withtriethanolamine at pH 8.2 (1:10 soil-to-extractant ratio), andCa, Mg, K and Na in extracts were analysed by flameatomic absorption spectrometry (AAS) (Soil Survey Labo-ratory Staff 1996). Then, the soil was saturated with 50 mMMgSO4. Magnesium remaining in the solution was mea-sured and correlated to the cation exchange capacity (CEC).The exchangeable sodium percentage (ESP) was calculatedas:

ESP ¼ Exchangeable sodium� 100=ΣEBC

Available Cu and Zn were extracted with 5.0 mM DTPA,0.01 M CaCl2 and 0.1 M triethanolamine at pH 7.3 anddetermined by flame AAS (Lindsay and Norwell 1978).

Soil microbiological analyses

The following analyses were performed on moist sampleswithin 15–20 days from soil sampling. Soil respiration wasdetermined as the CO2–C evolved and cumulated after 1 and10 days of incubation at 25±1°C according to Öhlinger(1995). A control with no soil was used to correct foratmospheric CO2. Metabolic quotient (qCO2) was calculatedas the ratio between CO2–C evolved after first day ofincubation and microbial biomass C (MBC) g−1 dry soil. Thehourly extra CO2–C cumulated during 6 h soil incubation at25°C after glucose amendment (4 mg glucose g−1 soil) was

considered as the substrate-induced respiration (SIR), asreported by Anderson and Domsch (1978).

Soil microbial biomass C (MBC) and N (MBN) contentswere determined by the fumigation–extraction methoddescribed by Vance et al. (1987) and Brookes et al.(1985), respectively, by considering the extra flushes of0.5 M K2SO4-extractable organic C and total N followingCHCl3 fumigation multiplied by 2.64 (KEC) and 2.22 (KEN)as conversion factors, respectively. The total extractable C(dichromate oxidation, Extr-C) and N (Kjeldahl method,Extr-N) were thus determined in the extracts of unfumi-gated soils (1 g soil:4 mL 0.5 M K2SO4). The microbialquotient values (MQC and MQN) were the percentages ofTOC and total N present as MBC and MBN, respectively.

Microbial adenosine triphosphate (ATP) was extractedfrom 0.5 g fresh soil according to Ciardi and Nannipieri(1990) and determined by the luciferin–luciferase assay(Lumac, Schaesberg, The Netherlands). An internal ATPstandard was added to soil to consider the adsorption ofATP by soil colloids.

Exchangeable NH4+ and NO3

− were extracted by 0.5 MK2SO4 (soil/extractant ratio 1:4, w/v). Ammonium wasdetermined by the indophenol blue colorimetric method andNO3

− after reduction to NO2− with Cu–Cd by the modified

Griess–Ilosvay method (Keeney and Nelson 1982).Arylsulphatase (E.C. 3.1.6.1), β-glucosidase (E.C.

3.2.1.21), phosphatase (E.C. 3.1.3.2) and invertase activi-ties (E.C. 3.2.1.26) were determined as described byGianfreda et al. (2005). Urease activity (E.C. 3.5.1.5) wasdetermined as described by Kandeler and Gerber (1988).Dehydrogenase (E.C. 1.1.) activity was measured accordingto Trevors (1984), while o-diphenol oxidase activity (E.C.1.10.3.1) was determined as described by Perucci et al.(2000). One unit (U) of enzyme activity was defined aseither the micromoles or micrograms of substrate hydro-lyzed at 30°C h−1 by 1 g of dry soil. The spontaneous orabiotic transformation (auto-hydrolysis) of substrates wasassessed by control tests performed with autoclaved (1 h at121°C) soils. Equal activity values were obtained withoutand with toluene (0.1 mL g−1 soil), thus ruling out anygrowth of microbial cells during the assays. Each enzymeassay was performed in triplicate.

Statistical analysis

Results are arithmetic means of five soil samples and areexpressed on an oven-dry basis (105°C). Two-way ANOVAwas performed with agricultural management (intensive,IA, and sustainable, SA) and time (sampling year 2005,2006 and 2007) as factors. Significant statistical differencesof all variables between IA and SA at the same year and atthe same agricultural management among years wereestablished by LSD test at P<0.05. Residual maximum

Biol Fertil Soils (2010) 46:481–489 483 Author's personal copy

likelihood variance components were also performed todetermine which of the two factors or their interactionaccounted for the majority of the variation in each of themeasured variable. All statistical analyses were carried outusing SPSS 13.0 for Windows.

Results

Soil physical and chemical properties

Soil bulk density and particle size distribution were onlydetermined at the first sampling (April 2005) since bothvariables were expected to be relatively stable through the3-year observation period. Bulk density of IA soil wassignificantly (P<0.05) higher than in SA (1.74±0.12 vs.1.40±0.11 kg dm−3) soil, whereas both soils had a clayloam texture.

Both actual (pH in water) and potential (pH in KCl) soilacidities were 0.6 to 0.9 units lower in IA than SA soil at anysampling (Table 1), while in both managements, the overallmean (n=15) exchangeable acidity (proportional to differ-ence between pH in water and pH in KCl) was lower thanactual acidity by one pH unit (pH in water 7.05 vs. 7.72 andpH in KCl 6.06 vs. 6.79 in soils IA and SA, respectively).

Two-way ANOVA showed that agricultural managementaffected (at least P<0.01) all the chemical variables, exceptexchangeable Mg2+ (Table 1). Sampling year affectedseveral soil variables, including exchangeable Mg2+ andNa+, ESP and EC (at least P<0.01) whose changes throughthe 3-year period could reflect changes in quality ofirrigation water, although it was the same, also quantita-tively, between IA and SA soils.

SA management significantly increased soil organiccarbon (TOC) content at any sampling (Table 1); averageTOC increased from 10.6 g kg−1 in the IA soil to 18.3 gkg−1 in SA soil (73% increase). Total N also increased butnot at the first sampling (overall mean from 1.6 g kg−1 inIA soil to 2.4 g kg−1 in the SA soil, a 50% increase).

The clay content was comparable in both managements,and as expected, the CEC trend was parallel to that of TOC.However, the Ca2+ saturation degree of the complexexchange was on average higher in SA than IA soil (overallmean 76% vs. 56%, respectively), despite the very lowamount of total soil carbonates (data not reported) of bothsoils. Moreover, the mean ESP constituted 5.9% of theEBC in IA vs. 1.5% in SA soil (Table 1).

EC was two to three times lower in SA than IA soilthrough the 3-year period (on average, 0.56 vs.1.56 dS m−1, respectively). DTPA-extractable Cu and Zncontents were also much lower in SA than IA soil at anysampling year, with average Cu and Zn contents twice andthree times as low, respectively (Table 1).

Soil microbiological properties

Most soil microbiological properties, much lower under IAthan SA soil, were significantly affected (P<0.001) byagricultural management, with the exclusion of K2SO4-extractable total N (Extr-N), phosphatase and o-diphenoloxidase activities (Tables 2, 3 and 4). With regard to MBC,SIR and qCO2 (the amount of CO2–C evolved per unit ofMBC during the first day of incubation), mean reductionunder IA ranged from 76% to 46%, respectively, whileMBN, ATP, MQC, MQN and CO2 at 1 and 10 days valueswere, on average, 71%, 65%, 60%, 57%, 56% and 51%lower, respectively (Table 2). Also enzyme activities werelower under IA management (on average, arylsulphatase,urease, invertase, β-glucosidase and dehydrogenase by85%, 81%, 76%, 63% and 50%, respectively) (Table 3).Interestingly, the three latter enzymes are linked to the Ccycle, whereas the two most lowered enzyme activities arerelated to S and N, i.e. two nutrients added by inorganicfertilisation.

The effect of sampling year only was not significant forMBC, MQC, Extr-C, arylsulphatase and phosphataseactivities (Tables 2, 3 and 4). The two variables mostlyaffected by time were Extr-N and ammonium (91% and84%, respectively, of variance explained).

As usual under Mediterranean conditions, the dynamicsof inorganic N was dominated by nitrate since it accountedfor at least 73% of the whole mineral N during the 3-yearperiod. Nitrate, mineral N and Extr-C were decreased bySA compared to IA (Table 4). Interestingly, all thesevariables are related to the available nutrient pool, andamong variables linked to C and N mineralisation–immobilisation processes, only Extr-N did not respond toagricultural management. However, by subtracting ex-changeable ammonium from Extr-N, the resulting organicExtr-Org N was significantly affected (Table 4). Theseresults once again confirm that reversing IA to SA favouredthe immobilisation of C and N into soil organic matter.

Inhibition of microbiological properties such as SIR,β-glucosidase and urease activities by increased sodicitymight be deduced as significant negative correlationsbetween each of these properties, and both exchangeableNa+ and ESP occurred in IA soil, where both exchangeableNa+ and ESP were higher, but not in SA soil (with SIR, r=−0.69 and −0.64; with β-glucosidase, r=−0.68 and −0.56;with urease, r=−0.73 and −0.61, at least P<0.05, n=15 inall cases).

Content of ATP in SA soil significantly correlated with10 variables (MBN, Extr-C, Extr-N, β-glucosidase, phos-phatase, invertase, o-diphenol oxidase and urease activities,exchangeable ammonium and mineral N (at least P<0.05,n=15)), whereas in IA soil, it significantly correlated withonly five variables (MBN, β-glucosidase activity, ex-

484 Biol Fertil Soils (2010) 46:481–489 Author's personal copy

Tab

le1

Phy

sico-chemical

prop

ertiesof

soilsamples

(n=5)

from

intensiveagricultu

re(IA)andsustainableagricultu

re(SA)plots

Sam

pling

pHTOC

Total

NTOC/Total

NCEC

Exchang

eablecatio

nsESP

EC

Cu

Zn

H2O

KCl

(gkg

−1)

Ca2

+Mg2

+

(cmol

(+)kg

−1)

Na+

K+

(%)

(dSm

−1)

(mgkg

−1)

IA_2

005

6.9Ab

5.8Ab

10.1

Ab

1.6Aa

6.5Aa

25.2

Ab

16.4

Ab

6.2Aa

1.3Ab

0.9Aa

5.1Ab

0.9Ab

5.1Ab

13.7

Ab

IA_2

006

7.1Aa

6.1Ab

10.7

Ab

1.6Ab

6.8Aa

25.4

Ab

14.0ABb

5.0Ba

2.2Bb

0.8Ab

8.6Bb

1.9Bb

9.2Bb

9.3Ab

IA_2

007

7.1Ab

6.3Ab

10.9

Ab

1.6Ab

7.0Aa

26.1

Ab

12.0

Bb

4.4Ba

1.0Ab

1.0Ab

3.9Ab

1.9Bb

10.9

Bb

17.7

Ab

SA_2

005

7.7Aa

6.7Aa

17.0

Aa

1.9Aa

8.7Aa

30.9

Aa

23.4

Aa

6.5Aa

0.8Aa

0.5Aa

2.5Aa

0.4Aa

2.6Aa

3.8Aa

SA_2

006

7.7Aa

6.8Aa

18.5

Aa

2.5Ba

7.5ABa

30.5

Aa

22.8

Aa

3.6Ba

0.3Ba

0.3ABa

0.9Ba

0.7Ba

5.1Ba

3.5Aa

SA_2

007

7.8Aa

6.8Aa

19.3

Aa

2.7Ba

7.5Ba

30.7

Aa

23.5

Aa

3.9Ba

0.3Ba

0.2Ba

1.0Ba

0.7Ba

4.1Ca

4.1Aa

Exp

lained

variance

(%)

Managem

ent

65.4**

*69

.5**

*86

.4**

*62

.2**

*20

.2*

72.5**

*83

.2**

*NS

54.5**

*66

.7**

*58

.6**

*61

.5**

*59

.9**

*70

.9**

*

Year

NS

NS

NS

6.3*

NS

NS

NS

50.2**

*8.5*

**NS

6.9*

*15

.3**

*23

.6**

*6.2*

M*Y

NS

NS

NS

13.4*

NS

NS

NS

NS

27.9**

*NS

22.2**

*10

.0*

14.0**

*8.6*

The

micronu

trientsCuandZnwereextractedby

DTPA

.Datawereanalysed

bytwo-way

ANOVA

(managem

entandsamplingyear

asfactors).Different

lowercase

letters

indicate

sign

ificant

differences(P

<0.05)betweenmanagem

entsat

thesameyear.Different

capitalletters

indicate

significantdifferences(P

<0.05

)am

ongyearswith

inthesamemanagem

ent

TOC

totalorganicC;CECcatio

nexchange

capacity;Ca,

Mg,

KandNaexchangeable

base

catio

ns;ESP

exchangeable

sodium

percentage;ECelectrical

cond

uctiv

ity;N.S.no

tsign

ificant

*P<0.05

;**

P<0.01

;**

*P<0.00

1

Biol Fertil Soils (2010) 46:481–489 485 Author's personal copy

changeable ammonium and CO2 evolved after 1 day ofincubation and dehydrogenase activity). Therefore, as theATP content may be assumed as an integrated indicator ofboth microbial biomass and activity (Nannipieri et al.1990), reversing IA management to SA once again seemedto indicated the soil quality improvement.

Discussion

IA practices based on the frequent soil tillage, long-terminorganic fertilisation and irrigation with moderately salt-affected waters decreased physical properties, with aremarkable reduction in organic matter content, and

Table 3 Soil enzyme activities (amount of hourly transformed substrate) of soil samples (n=5) from intensive agriculture (IA) and sustainableagriculture (SA) plots

Sampling Arylsulphatase(μmol g−1 h−1)

β-glucosidase(μg g−1 h−1)

Phosphatase Invertase Phenoloxidase Dehydrogenase Urease

IA_2005 0.03 Ab 0.7 Ab 1.3 Aa 1.2 Ab 27.2 Aa 0.8 Ab 7.1 Ab

IA_2006 0.03 Ab 0.4 Bb 1.2 Aa 3.3 Bb 27.2 Aa 1.0 Ab 3.1 Bb

IA_2007 0.05 Ab 0.8 Ab 1.0 Ab 3.3 Bb 40.6 Bb 1.2 Bb 9.7 Cb

SA_2005 0.2 Aa 1.7 Aa 0.9 Aa 6.8 Aa 32.6 Aa 1.5 Aa 46.5 Aa

SA_2006 0.3 Aa 1.5 Aa 1.1 ABa 11.9 Ba 31.9 Aa 2.4 Ba 7.6 Ba

SA_2007 0.3 Aa 2.1 Ba 1.3 Aa 13.5 Ba 38.0 Ba 1.9 ABa 52.0 Aa

Explained variance (%)

Management 92.2*** 84.4*** NS 78.0*** NS 67.9*** 47.9***

Year NS 9.2*** NS 12.8*** 45.4*** 10.0** 21.3***

M * Y NS NS NS 6.1*** NS 9.2* 24.6***

Data were analysed by two-way ANOVA (management and sampling year as factors). Different lowercase letters indicate significant difference (P<0.05) between managements at the same year. Different capital letters indicate significant differences (P<0.05) among years within the samemanagement

N.S. not significant

*P<0.05; **P<0.01; ***P<0.001

Table 2 Soil microbial properties of soil samples (n=5) from intensive agriculture (IA) and sustainable agriculture (SA) plots

Sampling MBC(µg g−1)

MBN(µg g−1)

ATP(µg g−1)

MQC(%)

MQN(%)

CO2 1d(μg CO2–C g−1)

CO2 10d(μg CO2–C g−1)

SIR(μg CO2–C g−1)

qCO2

(μg CO2–C μg−1

MBC d−1)

IA_2005 144.7 Ab 5.3 Ab 0.05 Ab 1.4 Aa 0.3 Ab 22.3 Ab 120.3 Ab 37.7 Ab 0.16 Aa

IA_2006 62.0 Bb 14.0 Ab 0.01 Ab 0.6 Bb 0.9 Ab 29.8 Ab 103.6 Ab 16.4 Bb 0.48 Bb

IA_2007 113.8 Ab 40.8 Bb 0.19 Bb 1.0 ABb 2.6 Ba 50.5 Bb 133.4 Ab 40.8 Aa 0.44 Bb

SA_2005 406.0 Aa 69.5 Aa 0.12 Aa 2.4 Aa 3.6 Aa 90.6 Aa 264.9 Aa 66.5 Aa 0.22 Aa

SA_2006 525.6 Aa 40.0 Aa 0.06 Aa 2.8 Aa 1.6 Ba 58.1 Ba 218.0 Ba 56.1 ABa 0.11 Ba

SA_2007 420.8 Aa 95.2 Ba 0.61 Ba 2.2 Aa 3.5 Aa 84.3 Aa 246.4 Aa 50.3 Ba 0.20 Aa

Explained variance (%)

Management 83.8*** 70.8*** 18.9*** 69.4*** 48.0*** 68.5*** 78.1*** 55.4*** 26.1***

Year NS 17.4*** 49.2*** NS 17.9*** 9.4*** N.S. 8.6* 6.2*

M * Y 5.6* NS 25.9*** 12.6* 24.9*** 15.9*** N.S. 15.5* 42.9***

The metabolic quotient (qCO2) corresponds to CO2 1d/MBC. Data were analysed by two-way ANOVA (management and sampling year asfactors). Different lowercase letters indicate significant difference (P<0.05) between managements at the same year. Different capital lettersindicate significant differences (P<0.05) among years within the same management

MBC microbial biomass C; MBN microbial biomass N; MQC and MQN microbial quotients on C and N basis, respectively; CO2 1d and CO2 10dCO2 cumulated after 1 and 10 days, respectively, of incubation at 25°C and 50% WHC; ATP adenosine triphosphate; SIR substrate (glucose)induced respiration (see text for details); N.S. not significant

*P<0.05; **P<0.01; ***P<0.001

486 Biol Fertil Soils (2010) 46:481–489 Author's personal copy

decreased most of the measured microbiological properties.As outlined by Speir and Ross (2002), intensive croppingregime can cause soil degradation as a consequence of lossof organic matter and structural integrity. Once theagricultural regime was reversed to a more sustainableone, SOM recovered, and this improved soil quality asshown by microbiological properties such as the metabolicquotient (qCO2) lower under SA than IA, and MQC andMQN, higher under SA than IA soil, regardless of samplingyear (Laudicina et al. 2009).

The increase in DTPA-extractable Cu and Zn byintensive inorganic fertilisation was probably due to thehigher dissolution of soil minerals caused by the formationof sulphuric and nitric acids (McGahan et al. 2003) withlower soil pH in IA than SA soil. However, extra Cu andZn ions could also derive as impurities from the addedinorganic fertilizers.

Soils are usually considered saline when EC of saturatedsoil paste extract is higher than 4 dS m−1 and sodic whenESP is higher than 15% (Rowell 1994). Although EC of IAsoil never exceeded 1.9 dS m−1, its sodic charactersignificantly increased if compared to SA soil, and thismay partially explain the enhanced soil bulk density(1.74 kg dm−3) as the result of the heavier structure inducedby the Na+-mediated collapse of clay particle aggregates.The increase in EC values of IA soil could have partiallyresulted from the long-term mineral fertilisation.

Reversing IA management to SA management almostdoubled the SOM and, as expected, activated mostmicrobiological variables linked to soil C and N cycles.However, most of microbiological properties of IA soil,

compared to SA soil, decreased on average more than theTOC did, probably due to the presence of inhibitory factorssuch as the higher soil solution osmotic pressure andsodicity, as shown by the higher EC, ESP and exchangeableNa+ values in IA than SA soil. These salinity and sodicityincreases were likely induced by the long-term mineralfertilisation, as irrigation waters were qualitatively andquantitatively the same between IA and SA soils. Negativelinear relationships between soil osmotic stress and micro-biological activities were obtained by Rietz and Haynes(2003) in a similar soil study, but contrary to our results,soil basal respiration did not decrease by increasing salinity.However, Rietz and Haynes (2003) did not report theduration of the salinity stress. The likely long-term decreasein TOC content of IA soil may have synergically acted withinhibition by moderate salinity and sodicity, and the wholeimpact on soil microbial communities could be similar tothat observed at high salinities (Garcia and Hernandez1996; Pankhurst et al. 2001).

All the carbon measured in extractable C pool (Extr-Cand dichromate oxidation) was organic, whereas the Nwithin the K2SO4-extractable N pool (Extr-N, Kjeldahlmethod) included extractable organic N and NH4

+–N butnot nitrate. Therefore, the Extr-C/Extr-Org N ratio, whereExtr-Org N is the Extr-N minus the K2SO4-exchangeableNH4

+, may represent the C/N ratio of soluble organicsubstrates (e.g. amino acids) produced by mineralisationand available to direct assimilation (Barraclough 1997).These ratios indicated the presence of amino acids in theSA soil (Table 4), while they were higher than C/N ratio ofamino acids in the IA soil. Usually, the higher the quality of

Table 4 Soil extractable C and N fractions (1 g soil:4 mL 0.5 M K2SO4) and related ratios from intensive agriculture (IA) and sustainableagriculture (SA) plots

Sampling Extr-C(µg g−1)

Extr-N(µg g−1)

NH4+–N

(µg g−1)NO3

−–N(µg g−1)

Inorganic N Extr-Org N Extr-C/Extr-Org N CO2 10d/Inorganic N

IA_2005 77.9 Ab 11.0 Aa 1.0 Aa 81.0 Ab 82.0 Ab 10.0 Aa 8.5 Aa 10.7 Ab

IA_2006 93.7 Bb 6.2 Bb 1.3 Aa 27.0 Bb 28.2 Bb 4.9 Bb 20.0 Bb 14.6 Ab

IA_2007 68.9 Aa 21.9 Ca 11.0 Bb 39.9 Bb 50.9 Cb 10.9 Ab 6.4 Aa 4.1 Bb

SA_2005 60.7 Aa 11.7 Aa 0.2 Aa 5.9 Aa 6.1 Aa 11.5 Aa 5.7 Aa 24.6 Aa

SA_2006 26.0 Ba 7.6 Ba 0.8 Aa 21.0 Ba 21.8 Ba 6.8 Ba 3.8 Ba 26.5 Aa

SA_2007 67.3 Aa 19.9 Ca 7.0 Ba 19.3 Ba 26.3 Ba 12.9 Aa 5.2 ABa 9.2 Ba

Explained variance (%)

Management 37.8*** NS 5.6*** 40.4*** 43.2*** 8.4* 30.5*** 68.6***

Year NS 90.6*** 84.0*** 6.4*** 5.8** 63.2*** 13.6*** 18.5***

M * Y 53.3*** NS 6.3** 45.9*** 43.1*** NS 45.4*** N.S.

Extr-Org N is Extr-N minus Inorganic N. Refer to Table 2 for the meaning of CO2 10d. Data were analysed by two-way ANOVA (managementand sampling year as factors). Different lowercase letters indicate significant difference (P<0.05) between managements at the same year.Different capital letters indicate significant differences (P<0.05) among years within the same management

Extr-C and Extr-N extractable total C and N, respectively; Inorganic N extractable ammonium-N plus nitrate N; N.S. not significant

*P<0.05; **P<0.01; ***P<0.001

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an organic substrate available to soil microorganisms, thelower its C/N ratio (Fog 1988). On the other hand,inorganic N and Extr-C, i.e. N and C forms readilyavailable too, were higher in soil IA than SA (Table 4).Thus, factors which involve not only the mass of readilyavailable C and N but also the substrate quality help toexplain the highly depressed microbiological properties inIA soil.

Due to the high percentage of available C lost as respiredCO2, C deficiency in soil may usually appear well before Ndeficiency (Hart et al. 1994; Gregorich et al. 2000). Thefact that, the 10 day-evolved CO2–C-to-inorganic N ratio, apossible indicator of potentially mineralisable C vs.inorganic N readily available to soil microorganisms, wasalways much higher in SA than IA soil (Table 4, P<0.05),it could indirectly indicate that higher C mineralisation,associated with greater microbial biomass N immobilisa-tion, occurred in SA soil, while in IA soil, the sameprocesses were lower. Therefore, the above ratio was foundvery sensitive to agriculture management.

The increase of organic matter in the SA soil, besidesimprovement of physical properties, increased enzymeacitivities because these properties are usually correlatedwith the soil organic matter content (Speir and Ross 2002).These increases in enzyme activities probably acceleratedthe turnover of the main nutrients such as C, N, P and S.Correspondingly, the decreases in most of enzyme activitiesin IA soil, compared with SA soil, were higher than thedecreases in TOC. The direct effect of osmotic potentialand specific ions on enzyme activities cannot be excluded.Indeed, salts may influence the catalytic behaviour ofenzymes by direct activation or inactivation or indirectlyby decreasing the solubility of the enzymatic moleculesthrough a “salting-out” effect (Frankenberger and Bingham1982). A cumulative inhibition by moderately salt-affectedirrigation and long-term inorganic fertilisation on enzymeactivities in IA soil can also be possible. However,phosphatase and o-phenoloxidase activities were not affect-ed by the agricultural management, and this indicates thatthe extent of the inhibition by osmotic stress and/or increasein TOC is enzyme specific.

Although no direct evidence of a back-inhibitory effectof sulphate on soil arylsulphatase activity has beenprovided (Al-Khafaji and Tabatabai 1979), the decline ofarylsulphatase activity in the IA soil occurred, whereas theconcentration of sulphate in soil solution increased due tothe long-term ammonium sulphate fertilisation. Arylsulpha-tase activity has been barely detected in gypsiferous soils,which naturally have an excess of sulphates (Garcia andHernandez 1997).

Both β-glucosidase and urease have been shown to bevery sensitive indicators of changes in several soil-functioning-related properties such as organic matter, active

microbial population and pH (Bandick and Dick 1999;Saviozzi et al. 2001). Furthermore, the activity of β-glucosidase has often been considered in most empiricalrelationships proposed as indexes of soil biochemicalquality (Nannipieri et al. 2002; Gianfreda and Ruggiero2006). In a similar study, a numerical index developed bymeans of canonical discriminant analysis and including theactivity of both β-glucosidase and urease well discriminat-ed between altered and control soils, comparable to our IAand SA soils, respectively (Puglisi et al. 2006).

Decreases in microbial indicators similar to thoseobserved in our study were generally reported by Garciaand Hernandez (1997) in soils from a semiarid SpanishMediterranean area subject to a high degree of erosion andwith the soil TOC content about 65% lower than theaverage TOC content of undisturbed natural soils in thesame area.

Conclusions

The soil organic matter content in SA soil increased due tothe farmyard manure incorporation and reduced tillage. Soilsalinity and sodicity were always higher in IA than SA soil(although not critically high) likely due to the intensiveinorganic fertilisation as irrigation waters for the two soilswere qualitatively and quantitatively the same. Thus, wesuggest that the cumulative small but long-term saline(osmotic) and sodic (dispersing) effects in IA soil decreasedthe microbial variables more than TOC and increased soilbulk density.

Significant decreases in SOM under Mediterraneanclimates seem to make soil functions and overall biologicalfertility vulnerable to further intensive agriculture, especial-ly if combined to irrigation with even moderately salt-affected waters.

Acknowledgements This work was partially funded by the ItalianMinistery of Education, Universities and Research (MIUR) through the2-year programme “Potential use of biological indicators in monitoringsoil quality for a sustainable environment”, which started in 1999.

References

Al-Khafaji AA, Tabatabai MA (1979) Effects of trace elements onarylsulfatase activity in soils. Soil Sci 127:129–133

Anderson JPE, Domsch KH (1978) A physiological method for thequantitative measurement of microbial biomass in soil. Soil BiolBiochem 10:215–221

Bandick AK, Dick RP (1999) Field management effects on soilenzyme activities. Soil Biol Biochem 31:1471–1479

Barraclough D (1997) The direct or MIT route for nitrogenimmobilization: a 15N mirror image study with leucine andglycine. Soil Biol Biochem 29:101–108

488 Biol Fertil Soils (2010) 46:481–489 Author's personal copy

Biederbeck VO, Campbell CA, Ukrainetz H, Curtin D, Bouman OT(1996) Soil microbial and biochemical properties after ten years offertilization with urea and anhydrous ammonia. Can J Soil Sci 76:7–14

Böhme L, Langer U, Böhme F (2005) Microbial biomass, enzymeactivities and microbial community structure in two Europeanlong-term field experiments. Agr Ecosyst Environ 109:141–152

Brookes PC, Kragt JF, Powlson DS, Jenkinson DS (1985) Chloroformfumigation and the release of soil nitrogen. Soil Biol Biochem17:831–835

Ciardi C, Nannipieri P (1990) A comparison of methods formeasuring ATP in soil. Soil Biol Biochem 22:725–727

Dick RP (1992) A review: long-term effects of agricultural systems onsoil biochemical and microbial parameters. Agric EcosystEnviron 40:25–36

Deng SP, Parham JA, Hattey JA, Babu D (2006) Animal manure andanhydrous ammonia amendment alter microbial carbon use effi-ciency, microbial biomass, and activities of dehydrogenase andamidohydrolases in semiarid agroecosystems. Appl Soil Ecol33:258–268

Doran JW, Sarrantonio M, Liebig MA (1996) Soil health andsustainability. Adv Agron 56:1–54

Elliott LF, Lynch JM, Papendick RI (1996) The microbial componentof soil quality. In: Stotzky G, Bollag J-M (eds) Soil biochemistry,vol. 9. Marcel Dekker, New York, pp 1–21

Fog K (1988) The effect of added nitrogen on the rate ofdecomposition of organic matter. Biol Rev 63:433–462

Frankenberger WT Jr, Bingham FT (1982) Influence of salinity on soilenzyme activities. Soil Sci Soc Am J 46:1173–1177

Garcia C, Hernandez T (1996) Influence of salinity on the biological andbiochemical activity of a calciorthid soil. Plant Soil 178:255–263

Garcia C, Hernandez T (1997) Biological and biochemical indicators inderelict soils subject to erosion. Soil Biol Biochem 29:171–177

Gianfreda L, Ruggiero P (2006) Enzyme activities in soil. In:Nannipieri P, Smalla K (eds) Nucleic acids and proteins in soil.Springer, Berlin, pp 257–311

Gianfreda L, Rao MA, Piotrowska A, Palumbo G, Colombo C (2005)Soil enzyme activities as affected by anthropogenic alterations:intensive agricultural practices and organic pollution. Sci TotalEnviron 341:265–279

Goyal S, Mishra MM, Dhankar SS, Kapor KK, Batra R (1993)Microbial biomass turnover and enzyme activities following theapplication of farmyard manure to field soil with and withoutprevious long-term applications. Biol Fertil Soils 15:60–64

Gregorich EG, Liang BC, Drury CF, Mackenzie AF, McGill WB(2000) Elucidation of the source and turnover of water solubleand microbial biomass carbon in agricultural soils. Soil BiolBiochem 32:581–587

Hart SC, Nason JE, Myrold DD, Perry DA (1994) Dynamics of grossnitrogen transformations in an old-growth forest: the carbonconnection. Ecology 75:880–891

Iovieno P, Morra L, Leone A, Pagano L, Alfani A (2009) Effect oforganic and mineral fertilizers on soil respiration and enzymeactivities of two Mediterranean horticultural soils. Biol FertilSoils 45:555–561

Izquierdo I, Caravaca F, Alguacil MM, Hernández G, Roldán A(2005) Use of microbiological indicators for evaluating successin soil restoration after revegetation of a mining area undersubtropical conditions. Appl Soil Ecol 30:3–10

Kandeler E, Gerber H (1988) Short-term assay of soil urease activityusing colorimetric determination of ammonium. Biol Fertil Soils6:68–72

Keeney DR, Nelson DW (1982) Nitrogen-inorganic forms. In: PageAL, Miller RH, Keeney DR (eds) Methods of soil analysis, part2. American Society of Agronomy, Madison, pp 643–698

Laudicina VA, Hurtado MD, Badalucco L, Delgado A, Palazzolo E,Panno M (2009) Soil chemical and biochemical properties of asalt-marsh alluvial Spanish area after long-term reclamation. BiolFertil Soils 45:691–700

Lindsay WL, Norwell WA (1978) Development of a DTPA test forzinc, iron, manganese, and copper. Soil Sci Soc Am J 42:421–428

McGahan DG, Southard RJ, Zasoski RJ (2003) Mineralogicalcomparison of agriculturally acidified and naturally acidic soils.Geoderma 114:355–368

Nannipieri P, Grego S, Ceccanti B (1990) Ecological significance ofthe biological activity in soil. In: Stotzky G, Bollag J-M (eds)Soil biochemistry, vol. 6. Marcel Dekker, New York, pp 293–355

Nannipieri P, Kandeler E, Ruggiero P (2002) Enzyme activities andmicrobiological and biochemical processes in soil. In: Burns RG,Dick RP (eds) Enzymes in the environment. Activity, ecologyand applications. Marcel Dekker, New York, pp 1–33

Nannipieri P, Ascher J, Ceccherini MT, Landi L, Pietramellara G,Renella G (2003) Microbial diversity and soil functions. Eur JSoil Sci 54:665–670

Öhlinger R (1995) Soil respiration by titration. In: Schinner F,Öhlinger R, Kandeler E, Margesin R (eds) Methods in soilbiology. Springer, Berlin, pp 95–98

Pankhurst CE, Yu S, Hawke B, Harch BD (2001) Capacity of fattyacid profiles and substrate utilization patterns to describe differ-ences in soil microbial communities associated with increasedsalinity or alkalinity at three locations in South Australia. BiolFertil Soils 33:204–217

Perucci P, Casucci C, Dumontet S (2000) An improved method toevaluate the o-diphenol oxidase activity of soil. Soil BiolBiochem 32:1927–1933

Puglisi E, Del Re AAM, Rao MA, Gianfreda L (2006) Developmentand validation of numerical indexes integrating enzyme activitiesof soils. Soil Biol Biochem 38:1673–1681

Rietz DN, Haynes RJ (2003) Effects of irrigation-induced salinity andsodicity on soil microbial activity. Soil Biol Biochem 35:845–854

Rowell DL (1994) Soil science: methods & applications. LongmanScientific & Technical, Essex, p 350

Saviozzi A, Levi-Minzi R, Cardelli R, Riffaldi R (2001) A comparisonof soil quality in adjacent cultivated, forest and native grasslandssoils. Plant Soil 233:251–259

Simek M, Hopkins DW, Kalcik J, Picek T, Santruckova H, Stana J,Travnik K (1999) Biological and chemical properties of arablesoils affected by long-term organic and inorganic fertilizerapplications. Biol Fertil Soils 29:300–308

Soil Survey Laboratory Staff (1996) Soil survey laboratory methodsmanual. Soil Survey Investigations Rep. 42V. 3.0. USDA-NRCS.U.S. Gov. Print. Office, Washington, DC

Soil Survey Staff (2006) Keys to soil taxonomy, 10th edn. USDA-Natural Resources Conservation Service, Washington

Speir TW, Ross DJ (2002) Hydrolytic enzyme activities to assess soildegradation and recovery. In: Burns RG, Dick RP (eds) Enzymesin the environment. Activity, ecology and applications. MarcelDekker, New York, pp 407–431

Trevors JT (1984) Dehydrogenase in soil: a comparison between theINT and TTC assay. Soil Biol Biochem 16:673–674

Vance ED, Brookes PC, Jenkinson DS (1987) An extraction methodfor measuring soil microbial biomass C. Soil Biol Biochem19:703–707

van Diepeningen AD, de Vos OJ, Korthals GW, van Bruggen AHC(2006) Effects of organic versus conventional management onchemical and biological parameters in agricultural soils. ApplSoil Ecol 31:120–135

Biol Fertil Soils (2010) 46:481–489 489 Author's personal copy