Research Article Chemical, Biochemical, and Microbiological Properties...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 496278 6 pageshttpdxdoiorg1011552013496278

Research ArticleChemical Biochemical and Microbiological Properties of Soilsfrom Abandoned and Extensively Cultivated Olive Orchards

A M Palese1 R Magno2 T Casacchia3 M Curci4 S Baronti2

F Miglietta2 C Crecchio4 C Xiloyannis1 and A Sofo5

1 Dipartimento delle Culture Europee e del Mediterraneo Architettura Ambiente Patrimoni Culturali (DiCEM)Universita degli Studi della Basilicata Via San Rocco 3 75100 Matera Italy

2 IBIMET-CNR Istituto di Biometeorologia Via G Caproni 8 50145 Firenze Italy3 CRA Centro di Ricerca per lrsquoOlivicoltura e lrsquoIndustria Olearia C da Li Rocchi-Vermicelli 87036 Rende Italy4Dipartimento di Biologia e Chimica Agroforestale e Ambientale Universita degli Studi di Bari Via Orabona 4 70126 Bari Italy5 Scuola di Scienze Agrarie Forestali Alimentari ed Ambientali Universita degli Studi della BasilicataViale dellrsquoAteneo Lucano 10 85100 Potenza Italy

Correspondence should be addressed to A M Palese assuntapaleseunibasit

Received 8 August 2013 Accepted 27 September 2013

Academic Editors L E Parent and C Sławinski

Copyright copy 2013 A M Palese et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The abandonment of olive orchards is a phenomenon of great importance triggered mainly by economic and social causes Theaim of this study was to investigate some chemical biochemical and microbiological properties in a soil of a southern olive groveabandoned for 25 years In order to define the effect of the long-term land abandonment on soil properties an adjacent olive grovemanaged according to extensive practices was taken as reference (essentially minimum tillage and no fertilization) Soil organicmatter total nitrogen and pH were significantly higher in the abandoned olive grove due to the absence of tillage and the naturalinputs of organic matter at high CN ratio which inter alia increased the number of cellulolytic bacteria and stimulated the activityof 120573-glucosidase an indicator of a more advanced stage of soil evolution The soil of the abandoned olive orchard showed a lowernumber of total bacteria and fungi and a lower microbial diversity measured by means of the Biolog method as a result of a sort ofspecialization trend towards low quality organic substrates From this point of view the extensive cultivation management seemedto not induce a disturbance to microbiological communities

1 Introduction

Olive trees have been cultivated for centuries becoming one ofthe most representative and stable crops with about 95 mil-lion of hectares [1] and an integral part of the Mediterraneanlandscape especially in the hilly and marginal parts of thisbasin [2] In spite of this the low yields compared withthe high costs of the mechanical operations (harvesting andpruning) the unfavourable oil price the competitionwith theforeign productions and the reduction of the EuropeanCom-munity financial aids determined in the last decades a pro-gressive olive orchard abandonment particularly inmarginaland inland agricultural areas of European producing coun-tries The consequence of this phenomenon can be a pos-itive restoration of natural vegetation or a negative soil

degradation due to the absence of conservation practicesespecially on slope lands [3 4]

In an abandoned olive orchard trees assume the originalbushy form canopies become dense and closed and pioneervegetation recolonizes free spaces according to ecologicalsuccessions that tend to arrive after long time to a naturalformation (ldquoclimaxrdquo) where soil and vegetation componentsare in equilibrium [2 5 6] During the transition of an olivegrove from a ldquodisturbedrdquo (cultivated) condition to a climaxphase we assume that soil properties progressively changeeven if very slowly as found in other similar agriculturalsystems [7 8]

Several studies were conducted on the effect of aban-donment of marginal agriculture in the Mediterranean areamost of them focused on the variability of physical and

2 The Scientific World Journal

chemical properties of soils during progressive vegetationcover changes [9ndash11] but few field researches include bio-chemical and microbiological factors as indicators of soilfertility restoration especially for olive orchards [7 8 12ndash16] Therefore the present study aimed to investigate somechemical biochemical and microbiological soil parametersof an olive orchard abandoned for 25 years In order toevaluate the effect of the long-term land abandonment on soilquality measurements were also carried out in a contiguoustraditional olive orchard cultivated according to extensiveagricultural practices that is low inputs of labour andmaterials [17]

2 Materials and Methods

21 The Experimental Olive Orchard The study was carriedout in a 2 ha rainfed olive orchard located on a flat land inthe rural area of Lucera (south-eastern Italy Puglia region41∘271015840383210158401015840N 15∘221015840137510158401015840E) Soil is a Vertisol classifiedas a Typic Calcixererts fine mixed thermic sandy clayloam (USDA soil taxonomy) Climate is typically Mediter-ranean characterized by hot and dry summer with a meanannual temperature of 157∘C and an average rainfall of583mmyearminus1

Olive trees (Olea europaea L cv Perenzana) were plantedin 1970 In 1985 34 of the orchard were completely aban-doned and its appearance has taken the form of Mediter-ranean coppice with shrubs herbs and weeds colonizing thespace between trees and rows (ldquoabandonedrdquo olive grove) Inthe ldquomanagedrdquo olive orchard pruning was made every twoyears and residues were burned far from the field Shallowtillage (15-cm depth) was carried out two times per year at thebeginning of Spring and in Autumn before harvest in orderto bury weeds and grasses During the interval between suchdates the soil remained covered by the spontaneous vegeta-tion No fertilizer application was performedThe trees in themanaged part of the field (220 trees haminus1) were constitutedfrom a single trunk (73) or from two trunks (26) while inthe abandoned part the main trunk was difficult to identifyas suckers were predominant (up to 11)

22 Soil Sampling Strategy and Chemical Analyses In July2010 three composite soil samples (each obtained on site byfour pooled cores of seven cm of diameter) were collectedfrom the top soil layers (0ndash20 and 20ndash40 cm) along theinterrows of both managed and abandoned olive orchardsSamples were stored at 4∘C in sterile plastic bags until thefollowing analyses

Soil subsamples were air dried and sieved at 2mm Onthese samples the following analyses were carried out accord-ing to the official methods of soil analysis [18] electricalconductivity and pH measured on a mixture of soil (10 g dryweight) and distilled water (25mL) and shaken for at least 2hours on a shaker at 40 rpm soil organicmatter (SOM) by thedichromate oxidation method total nitrogen by the Kjeldahlmethod available phosphorus by the Olsen method

Soil enzyme activities were measured on fresh soil sam-ples 120573-glucosidase activity was determined according toEivazi and Tabatabai [19] and expressed as mg p-nitrophenol

hminus1 gminus1 soilThe dehydrogenase assay was performed accord-ing to the method of Von Mercı and Schinner [20] andthe activity expressed as mg triphenylformazan hminus1 gminus1 soilFor cellulase measured by the method of Hope and Burns[21] the activity was expressed as g glucose hminus1 gminus1 soil Theenzyme activities were expressed as units per g of dry soil

23 Soil Microbial Counts and Microbial Community Meta-bolic Profiles (Biolog) Three replicates of 5 g subsamples(dry weight equivalent) of each fresh soil sample weresuspended in 45mL sterile 01 sodium pyrophosphate-onequarter strength Ringer solution and sonicated at 35 kHz for2min to disperse microbial cells Tenfold serial dilutions ofthe supernatants were made in a sterile Ringer solutionAliquots were spread plated in triplicate on 110 strengthTryptic Soy Agar (TSA) medium amended with cyclo-heximide 01mgmLminus1 for bacterial counting and inoculatedin Malt Extract Agar (MEA) medium implemented withstreptomycin 003mgmLminus1 and tetracycline 002mgmLminus1[22] in triplicate for fungal counting Cellulolytic bacteriawere spread and included between two double agar lay-ers using the following culture medium cellulose pow-der 5 g Lminus1 NH

4H2PO42 g Lminus1 KH

2PO406 g Lminus1 K

2HPO4

g Lminus1 MgSO4sdot7H2O 08 g Lminus1 tiamine 01mg Lminus1 adenine

4mg Lminus1 adenosine mg Lminus1 yeast extract 05 g Lminus1 and agar17 g Lminus1 Counting took place after suitable incubation period(72 h for bacteria and 120 h for fungi) at 28∘C

Sole carbon source utilization patterns of soil micro-bial communities also called community-level physiologicalprofiles (CLPPs) were assessed using the Biolog 96-wellEco-Microplates (AES Laboratoire France) containing 31different carbon sources three times replicated Data wereanalysed to determine metabolic diversity indices includingAverageWell ColourDevelopment (AWCD) (themean of theblanked absorbance values for all the substrates) Shannonrsquossubstrate diversity index (1198671015840) substrate evenness (119864 equi-tability of activities across all utilized substrates) and sub-strate richness (119878 the number of utilized substrates) accord-ing to Zak et al [23] and Sofo et al [24] The microplateswere incubated at 25∘C in the dark and colour develop-ment was measured as optical density (OD) every 24 hover a 144 h period using a Microplate E-Max Reader (Bio-Rad) with a E590 nm wavelength filter The substrate utiliza-tion profiles were analyzed on well absorbance values at the96 h observation period

24 Statistical Analysis The statistical analysis of data wascarried out using the SigmaStat 31 SPSS Inc software Two-way analysis of variance (ANOVA) of the soil parameterswas performed with orchard management and soil depth asfixed factors Means were separated according to Duncanrsquosmultiple comparison test at 119875 lt 005 and 119875 lt 001 Rela-tionships among soil properties were studied using Pearsoncorrelations The number of measured samples is specifiedthroughout the text and in the figure captions

The Scientific World Journal 3

Table 1 Two-way ANOVA analysis of chemical properties of the soils studied (average values n = 6)

Treatment SOM (g kgminus1) Total N (g kgminus1) CN Available P (mg kgminus1) pH Electric conductivity (120583S cmminus1)Management P = 0016 P lt 00001 ns P lt 00001 P = 00002 ns

Managed 277 b 13 b 122 a 560 a 79 a 804 aAbandoned 386 a 19 a 118 a 315 b 71 b 700 a

Soil depth ns ns ns ns P = 00085 ns0ndash20 cm 332 a 16 a 120 a 437 a 75 a 803 a20ndash40 cm 331 a 16 a 120 a 414 a 74 b 701 a

Management times soil depth ns ns ns ns P = 00017 nsns not significant mean values followed by different letters are significantly different between the two management systems

Table 2 Two-way ANOVA analysis of microbial counts and enzyme activities of the soils studied (average values n = 6)

Treatment Total bacteria(log CFU gminus1)

Total fungi(log CFU gminus1)

Cellulolytic bacteria(log CFU gminus1)

120573-glucosidase(units gminus1)

Dehydrogenase(units gminus1)

Cellulase(units gminus1)

Management P = 00004 P lt 00001 P lt 00001 P = 00004 ns nsManaged 94 a 80 a 24 b 165 b 893 a 136 aAbandoned 87 b 56 b 46 a 365 a 925 a 160 a

Soil depth P = 00038 ns ns ns P = 00176 ns0ndash20 cm 88 b 69 a 35 a 256 a 1057 a 147 a20ndash40 cm 93 a 68 a 35 a 275 a 760 b 148 a

Management times soil depth P = 00007 P = 00032 P = 00023 ns ns nsns not significant mean values followed by different letters are significantly different between the two management systems

3 Results and Discussion

Soil electrical conductivity did not differ between the twosystems whereas pH showed significantly higher values inthe managed orchard (Table 1) The lowering of pH in theabandoned grove could be attributed to the quality of theorganic material particularly rich in soil-acidifying com-pounds such as polyphenols and organic acids contained inolive leaves and fruits [25]

SOM is a fertility parameter that responds to changes insoil management in the long term [26] SOM of the aban-doned olive orchard was significantly higher than that foundin the managed treatment (Table 1) This increase was relatedto both the lack of soil disturbance by tillage [27] and thecontinuous natural inputs of organicmatter occurring during25 years of abandonment which provided the soil of carbonand energy sources [13] These inputs were derived fromolive trees and shrub-herbaceous plants (olive fruits senes-cent leaves shoots and branches other plant abovegroundbiomass roots root exudates) which settled widely inthe free spaces between the interrow areas and produced alow quality litter characterized by high content of lignin andpolyphenols or a low content in N (CN gt 25) [28] In anycase even the soil of themanaged olive showed a good level ofSOM due to both its pedologic origin (Vertisol a deep blackclay soil) [29] and to soil extensive management (minimumtillage andweed burial) Similarly Alvarez et al [3] found thatSoil Organic Carbon (SOC) content of organic olive groves(tilled once a year usually in spring or grazed at differentintensities) located in Southern Spain was relatively highcompared with the values reported for rainfed agriculturalsoils in the region (below 1) The authors also found that

SOC contents tended to be higher in undisturbed areas withnatural vegetation than in the abovementioned organic oliveorchards

The measured total nitrogen in the abandoned grove(19 g kgminus1 in the 0ndash40 cm soil layer) was higher (119875 lt0001) than the value observed in the managed system(13 g kgminus1) (Table 1) Furthermore a good correlation wasfound between organic matter and total nitrogen (119903 = 091119875 lt 0001)

No significant differences between the two managementsystems were found in the CN ratio which fluctuatedbetween 118 and 122 in the 0ndash40 cm soil layer (Table 1) A soilCN ratio of 10 is considered optimal for the best incorpora-tion rate of the organic matter into the soil profile [10]

The content of 119875Olsen in soil of managed olive orchard wassignificantly higher than in the abandoned treatment (119875 lt0001) (Table 1) Probably the phosphorus in plant residueswhich were buried three months before soil sampling (July2010) became available during their decomposition Never-theless 119875Olsen in the abandoned soil was enough to supportvegetation growth and development [3]

Total bacteria and fungi were more numerous in themanaged orchard than in the abandoned one (Table 2)

However the importance of these groups of microorgan-isms in terms of organic matter decomposition and otherbiochemical changes is linked to their relative abundance andfunctional diversity due to the metabolism and not to theirnumbers [30] In the present study the bacteria to fungi ratiowas higher in soil of the abandoned orchard (16 versus 12in the 0ndash40 cm soil layer) indicating that in this treatmentbacterial decomposition was favored over fungal decompo-sition For cellulolytic bacteria an opposite trend was found


0000010002000300040005000600

ManagedAbandoned

lowast

Polysaccharides and complex molecules

Cellulose

Emicellulose

Chitinlowast

Phosphorylatedcompounds

lowast

Organicacidslowast

lowast Aminoacids

lowast

Biogenicamines

(a)

ManagedAbandoned

0000010002000300040005000600

lowast


lowast

Cellulose

Emicellulose lowast

Chitinlowast


lowast

Organicacidslowast

lowastAminoacids

lowast

Biogenicamines

(b)

Figure 1 Radar diagrams of the average well colour development (AWCD) values for the eight main groups of the Biolog carbon compoundsin the 0ndash20 cm (a) and 20ndash40 cm (b) soil layer of managed (continuous line) and abandoned orchard (dashed line) The means (119899 = 9) withthe asterisk are significantly different between the two systems at 119875 lt 001

being higher in the abandoned grove (Table 2) This could beprobably due to the high inputs of cellulosicmaterial derivingfrom a more dense shrub vegetation occurring in the aban-doned olive grove It is well known that plant species (interms of quantity and quality) strongly affect the compositionof microbial communities during vegetation succession bymeans of rhizodeposition and the decay of litter and roots [1331]The change in soil energy pathways could determine as aconsequence a taxonomic shift in the composition activityand functional diversity of soil microbial biomass [32]

The extracellular soil enzyme 120573-glucosidase hydrolyzesorganic matter so releasing glycosidic residues such as glu-cose and galactose The activity of this enzyme is an excellentindicator of the degree of evolution and maturity of a soil Itincreases in the final stages of an ecological succession as itis related to biomass turnover and strongly depends on thesoil management adopted [14 19 33 34] In this study 120573-glucosidase activitywas significantly higher in the abandonedorchard (Table 2) which resemblesmore the later stages of anecological succession (climax) and was significantly relatedto SOM (119903 = 088 119875 lt 0001) In contrast dehydrogenasesisoforms are common to most organisms with a predom-inantly intracellular localization They are good indicatorsof the viability of bacterial populations and their oxidativemetabolism [20] In our case dehydrogenase activity did notdiffer statistically between the two treatments and appearedto be strongly influenced by soil depth (Table 2) Finallycellulases are a family of enzymes mainly produced by fungibacteria and protozoa belonging to the family of hydrolases

which catalyze the hydrolysis of 14-120573-D glycosidic bonds ofcellulose Their activity was not different from a statisticalpoint of view (Table 2)

The functional diversity of soil microbial communitiesestimated by the Biologmetabolic assay is based on the abilityof the microbial strains to oxidize different carbon sourcesand it has a high discriminating power among microbial soilcommunities [23]The community-level physiological profile(CLPP) obtained by this method was used to differentiatethe soil bacterial populations of the two orchards Data showthat among the indexes of microbial diversity examined Sand AWCD were significantly higher in the managed system(Table 3) indicating a higher bacterial functional diversityand complexity of this part of the grove

The 31 carbon substrates of the Biolog plates can bedivided into eight main groups (polysaccharides and com-plex molecules cellulose hemicellulose chitin phosphory-lated compounds organic acids aminoacids and biogenicamines) The radar diagrams of AWCD values of thesegroups are reported in Figure 1 As AWCD values providea measure of the cultural bacterial activity for each groupof compounds it is noteworthy that with the exception ofcellulose and hemicellulose in the 0ndash20 cm soil layer bacterialactivity due to substrate degradation was significantly higherin the managed orchard This is in accordance with thedifferences found in total bacteria and total AWCD whichwere significantly higher in the managed soil than in theabandoned one (Tables 1 and 3) Interestingly in the surfacesoil layer cellulose and emicellulose bacterial degradation


Table 3 Two-way ANOVA analysis of the indices used for theCommunity Level Physiological Profiling (CLPP Biologmethod) inthe studied soils (average values n = 9)

Treatment H1015840 S E AWCDManagement ns P = 00003 ns P = 00047

Managed 25 a 123 a 24 a 033 aAbandoned 21 a 86 b 22 a 019 b

Soil depth ns ns ns ns0ndash20 cm 24 a 106 a 24 a 027 a20ndash40 cm 23 a 105 a 23 a 025 a

Management times soil depth ns ns ns nsns not significant mean values followed by different letters are significantlydifferent between the two management systems H1015840 Shannonrsquos substratediversity index S substrate richness E substrate evenness AWCD averagewell colour development

did not show significant differences (Figure 1) and explainthe differences in cellulose activity between cultivated andabandoned orchards reported in Table 2

The higher bacterial functional diversity and complexityrecorded in the managed orchard could be due to the morebiodegradable substrates which were highly palatable tomicrobial communities In fact simple organic carbon suchas simple sugars and amino acids are quickly absorbed andprovide nutrients to microbes [35] Probably soil biota of thecultivated orchard were positively affected by the high litterquality (in terms of less phenolic substances and more nitro-gen) produced within the managed olive orchard (essentiallyplants belonging to Gramineae Composite and Legumi-nosae families) periodically buried into the soil by tillageand thus more easily decomposable By contrast the increaseof low quality organic inputs in the abandoned soil seemedto determine a bacterial specialization towards substratescharacterized by high CN ratio and low degradation rateresulting in a fewer ecological niches

4 Conclusions

Chemical changes occurred in the soil of the olive orchardafter 25 years from the abandonment were evident and tosome extent predictable due to the natural inputs of organicmatter and the absence of tillage Likewise the high 120573-glucosidase activity strongly related to soil organic matterplaced soil from abandoned grove in an advanced evolution-ary stage On the other hand the study of the carbon substrateutilization profiles performed by the Biolog method high-lighted unexpectedly a lower microbial diversity in the aban-doned orchard Taking into account this finding it would beadvisable to deepen such aspect by studying soil develop-ment in abandoned olive orchards using a chronosequenceapproach

Acknowledgment

The authors thank Professor Antonio Scopa from Universityof Basilicata for his constructive suggestions

References

[1] FAOSTAT 2012 httpfaostatfaoorg[2] A Loumou and C Giourga ldquoOlive groves lsquothe life and identity

of the Mediterraneanrsquordquo Agriculture and Human Values vol 20no 1 pp 87ndash95 2003

[3] S Alvarez M A Soriano B B Landa and J A Gomez ldquoSoilproperties in organic olive groves compared with that in naturalareas in a mountainous landscape in southern Spainrdquo Soil Useand Management vol 23 no 4 pp 404ndash416 2007

[4] M Koulouri and C Giourga ldquoLand abandonment and slopegradient as key factors of soil erosion inMediterranean terracedlandsrdquo Catena vol 69 no 3 pp 274ndash281 2007

[5] C Blasi R D Pietro and P Fortini ldquoA phytosociological analy-sis of abandoned terraced olive grove shrublands in the Tyrrhe-nian district of Central Italyrdquo Plant Biosystems vol 134 no3 pp 305ndash331 2000

[6] R Di Pietro and C Blasi ldquoA phytosociological analysis of aban-doned olive-grove grasslands of Ausoni mountains (Tyrrheniandistrict of Central Italy)rdquo Lazaroa vol 23 pp 73ndash93 2002

[7] P Zhang L Li G Pan and J Ren ldquoSoil quality changes inland degradation as indicated by soil chemical biochemical andmicrobiological properties in a karst area of southwest GuizhouChinardquo Environmental Geology vol 51 no 4 pp 609ndash619 2006

[8] R Zornoza J Mataix-Solera C Guerrero V Arcenegui and JMataix-Beneyto ldquoComparison of soil physical chemical andbiochemical properties among native forest maintained andabandoned almond orchards in mountainous areas of EasternSpainrdquo Arid Land Research and Management vol 23 no 4 pp267ndash282 2009

[9] C Kosmas S Gerontidis and M Marathianou ldquoThe effect ofland use change on soils and vegetation over various lithologicalformations on Lesvos (Greece)rdquoCatena vol 40 no 1 pp 51ndash682000

[10] G Dunjo G Pardini and M Gispert ldquoLand use change effectson abandoned terraced soils in aMediterranean catchment NESpainrdquo Catena vol 52 no 1 pp 23ndash37 2003

[11] H Garcıa D Tarrason M Mayol N Male-Bascompte and MRiba ldquoPatterns of variability in soil properties and vegetationcover following abandonment of olive groves in Catalonia (NESpain)rdquo Acta Oecologica vol 31 no 3 pp 316ndash324 2007

[12] A J Hernandez C Lacasta and J Pastor ldquoEffects of differentmanagement practices on soil conservation and soil water in arainfed olive orchardrdquo Agricultural Water Management vol 77no 1ndash3 pp 232ndash248 2005

[13] G Jia J Cao C Wang and G Wang ldquoMicrobial biomassand nutrients in soil at the different stages of secondary forestsuccession in Ziwulin northwest Chinardquo Forest Ecology andManagement vol 217 no 1 pp 117ndash125 2005

[14] E Benitez R Nogales M Campos and F Ruano ldquoBiochemicalvariability of olive-orchard soils under different managementsystemsrdquo Applied Soil Ecology vol 32 no 2 pp 221ndash231 2006

[15] B Wang G B Liu S Xue and B Zhu ldquoChanges in soilphysico-chemical and microbiological properties during nat-ural succession on abandoned farmland in the Loess PlateaurdquoEnvironmental Earth Sciences vol 62 no 5 pp 915ndash925 2011

[16] B Wang S Xue G B Liu G H Zhang G Li and Z P RenldquoChanges in soil nutrient and enzyme activities under differentvegetations in the Loess Plateau areaNorthwestChinardquoCatenavol 92 pp 186ndash195 2012


[17] F Duarte N Jones and L Fleskens ldquoTraditional olive orchardson sloping land sustainability or abandonmentrdquo Journal ofEnvironmental Management vol 89 no 2 pp 86ndash98 2008

[18] MiPAF and Italian Ministry for Agricultural and Forestry Poli-tics ldquoOfficial methods of soil chemical analysisrdquo Gazzetta Uffi-ciale Supplemento Ordinario 248 Istituto Poligrafico e Zeccadello Stato Rome Italy 1999

[19] F Eivazi and M A Tabatabai ldquoGlucosidases and galactosidasesin soilsrdquo Soil Biology and Biochemistry vol 20 no 5 pp 601ndash606 1988

[20] W von Mersi and F Schinner ldquoAn improved and accuratemethod for determining the dehydrogenase activity of soils withiodonitrotetrazolium chloriderdquoBiology and Fertility of Soils vol11 no 3 pp 216ndash220 1991

[21] C F A Hope and R G Burns ldquoActivity origins and location ofcellulases in a silt loam soilrdquo Biology and Fertility of Soils vol 5no 2 pp 164ndash170 1987

[22] H J Lorch G Benckiser and J C G Ottow ldquoBasic methodsfor counting microorganisms in soil and waterrdquo in Methods inApplied Soil Microbiology and Biochemistry K Aleph and PNannipieri Eds pp 146ndash161 Academic Press London UK1998

[23] J C Zak M R Willig D L Moorhead and H G WildmanldquoFunctional diversity of microbial communities a quantitativeapproachrdquo Soil Biology and Biochemistry vol 26 no 9 pp 1101ndash1110 1994

[24] A Sofo A M Palese T Casacchia et al ldquoGenetic functionalandmetabolic responses of soil microbiota in a sustainable oliveorchardrdquo Soil Science vol 175 no 2 pp 81ndash88 2010

[25] R Sansoucy XAlibes P Berge FMartilotti ANefzaoui andPZoıopoulos ldquoOlive by-products for animal feedrdquo FAO AnimalProduction and Health Paper 43 FAO Rome Italy 1985

[26] C Feller E BlanchartM Bernoux R Lal andRManlayc ldquoSoilfertility concepts over the past two centuries the importanceattributed to soil organic matter in developed and developingcountriesrdquoArchives of Agronomy and Soil Science vol 3 pp S3ndashS21 2012

[27] K Paustian H P Collins and E A Paul ldquoManagement controlson soil carbonrdquo in Soil Organic Matter in Temperate Agroe-cosystems Long-Term Experiments in North America E A PaulK Paustian E T Elliott and C V Cole Eds pp 15ndash49 CRCPress Boca Raton Fla USA 1997

[28] P Praveen-Kumar J C Tarafdar J Panwar and S Kathju ldquoArapidmethod for assessment of plant residue qualityrdquo Journal ofPlant Nutrition and Soil Science vol 166 no 5 pp 662ndash6662003

[29] Regione Puglia ldquoProgetto Acla 2mdashstudio per la caratteriz-zazione agronomica della regione Puglia e la classificazione delterritorio in funzione della potenzialita produttivardquo 2001 Pro-getto ACLA 2 POP Puglia 94ndash99 Sottoprogramma FEOGAhttpwwwcartograficopugliait

[30] F E Allison Soil OrganicMatter and Its Role inCrop ProductionDevelopments in Soil Science Elsevier Scientific London UK1973

[31] P Nannipieri J Ascher M T Ceccherini L Landi G Pietra-mellara andG Renella ldquoMicrobial diversity and soil functionsrdquoEuropean Journal of Soil Science vol 54 no 4 pp 655ndash6702003

[32] D A Wardle R D Bardgett J N Klironomos H Setala W Hvan der Putten and D H Wall ldquoEcological linkages betweenaboveground and belowground biotardquo Science vol 304 no5677 pp 1629ndash1633 2004

[33] EMadejon P Burgos R Lopez and F Cabrera ldquoSoil enzymaticresponse to addition of heavy metals with organic residuesrdquoBiology and Fertility of Soils vol 34 no 3 pp 144ndash150 2001

[34] C Crecchio M Curci A Pellegrino P Ricciuti N Tursi andP Ruggiero ldquoSoil microbial dynamics and genetic diversity insoil under monoculture wheat grown in different long-termmanagement systemsrdquo Soil Biology andBiochemistry vol 39 no6 pp 1391ndash1400 2007

[35] A R Maharning A A S Mills and S M Adl ldquoSoil com-munity changes during secondary succession to naturalizedgrasslandsrdquoApplied Soil Ecology vol 41 no 2 pp 137ndash147 2009

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ClimatologyJournal of


chemical properties of soils during progressive vegetationcover changes [9ndash11] but few field researches include bio-chemical and microbiological factors as indicators of soilfertility restoration especially for olive orchards [7 8 12ndash16] Therefore the present study aimed to investigate somechemical biochemical and microbiological soil parametersof an olive orchard abandoned for 25 years In order toevaluate the effect of the long-term land abandonment on soilquality measurements were also carried out in a contiguoustraditional olive orchard cultivated according to extensiveagricultural practices that is low inputs of labour andmaterials [17]

2 Materials and Methods

21 The Experimental Olive Orchard The study was carriedout in a 2 ha rainfed olive orchard located on a flat land inthe rural area of Lucera (south-eastern Italy Puglia region41∘271015840383210158401015840N 15∘221015840137510158401015840E) Soil is a Vertisol classifiedas a Typic Calcixererts fine mixed thermic sandy clayloam (USDA soil taxonomy) Climate is typically Mediter-ranean characterized by hot and dry summer with a meanannual temperature of 157∘C and an average rainfall of583mmyearminus1

Olive trees (Olea europaea L cv Perenzana) were plantedin 1970 In 1985 34 of the orchard were completely aban-doned and its appearance has taken the form of Mediter-ranean coppice with shrubs herbs and weeds colonizing thespace between trees and rows (ldquoabandonedrdquo olive grove) Inthe ldquomanagedrdquo olive orchard pruning was made every twoyears and residues were burned far from the field Shallowtillage (15-cm depth) was carried out two times per year at thebeginning of Spring and in Autumn before harvest in orderto bury weeds and grasses During the interval between suchdates the soil remained covered by the spontaneous vegeta-tion No fertilizer application was performedThe trees in themanaged part of the field (220 trees haminus1) were constitutedfrom a single trunk (73) or from two trunks (26) while inthe abandoned part the main trunk was difficult to identifyas suckers were predominant (up to 11)

22 Soil Sampling Strategy and Chemical Analyses In July2010 three composite soil samples (each obtained on site byfour pooled cores of seven cm of diameter) were collectedfrom the top soil layers (0ndash20 and 20ndash40 cm) along theinterrows of both managed and abandoned olive orchardsSamples were stored at 4∘C in sterile plastic bags until thefollowing analyses

Soil subsamples were air dried and sieved at 2mm Onthese samples the following analyses were carried out accord-ing to the official methods of soil analysis [18] electricalconductivity and pH measured on a mixture of soil (10 g dryweight) and distilled water (25mL) and shaken for at least 2hours on a shaker at 40 rpm soil organicmatter (SOM) by thedichromate oxidation method total nitrogen by the Kjeldahlmethod available phosphorus by the Olsen method

Soil enzyme activities were measured on fresh soil sam-ples 120573-glucosidase activity was determined according toEivazi and Tabatabai [19] and expressed as mg p-nitrophenol

hminus1 gminus1 soilThe dehydrogenase assay was performed accord-ing to the method of Von Mercı and Schinner [20] andthe activity expressed as mg triphenylformazan hminus1 gminus1 soilFor cellulase measured by the method of Hope and Burns[21] the activity was expressed as g glucose hminus1 gminus1 soil Theenzyme activities were expressed as units per g of dry soil

23 Soil Microbial Counts and Microbial Community Meta-bolic Profiles (Biolog) Three replicates of 5 g subsamples(dry weight equivalent) of each fresh soil sample weresuspended in 45mL sterile 01 sodium pyrophosphate-onequarter strength Ringer solution and sonicated at 35 kHz for2min to disperse microbial cells Tenfold serial dilutions ofthe supernatants were made in a sterile Ringer solutionAliquots were spread plated in triplicate on 110 strengthTryptic Soy Agar (TSA) medium amended with cyclo-heximide 01mgmLminus1 for bacterial counting and inoculatedin Malt Extract Agar (MEA) medium implemented withstreptomycin 003mgmLminus1 and tetracycline 002mgmLminus1[22] in triplicate for fungal counting Cellulolytic bacteriawere spread and included between two double agar lay-ers using the following culture medium cellulose pow-der 5 g Lminus1 NH

4H2PO42 g Lminus1 KH

2PO406 g Lminus1 K

2HPO4

g Lminus1 MgSO4sdot7H2O 08 g Lminus1 tiamine 01mg Lminus1 adenine

4mg Lminus1 adenosine mg Lminus1 yeast extract 05 g Lminus1 and agar17 g Lminus1 Counting took place after suitable incubation period(72 h for bacteria and 120 h for fungi) at 28∘C

Sole carbon source utilization patterns of soil micro-bial communities also called community-level physiologicalprofiles (CLPPs) were assessed using the Biolog 96-wellEco-Microplates (AES Laboratoire France) containing 31different carbon sources three times replicated Data wereanalysed to determine metabolic diversity indices includingAverageWell ColourDevelopment (AWCD) (themean of theblanked absorbance values for all the substrates) Shannonrsquossubstrate diversity index (1198671015840) substrate evenness (119864 equi-tability of activities across all utilized substrates) and sub-strate richness (119878 the number of utilized substrates) accord-ing to Zak et al [23] and Sofo et al [24] The microplateswere incubated at 25∘C in the dark and colour develop-ment was measured as optical density (OD) every 24 hover a 144 h period using a Microplate E-Max Reader (Bio-Rad) with a E590 nm wavelength filter The substrate utiliza-tion profiles were analyzed on well absorbance values at the96 h observation period

24 Statistical Analysis The statistical analysis of data wascarried out using the SigmaStat 31 SPSS Inc software Two-way analysis of variance (ANOVA) of the soil parameterswas performed with orchard management and soil depth asfixed factors Means were separated according to Duncanrsquosmultiple comparison test at 119875 lt 005 and 119875 lt 001 Rela-tionships among soil properties were studied using Pearsoncorrelations The number of measured samples is specifiedthroughout the text and in the figure captions



























0000010002000300040005000600

ManagedAbandoned

lowast


Cellulose

Emicellulose

Chitinlowast


lowast

Organicacidslowast

lowast Aminoacids

lowast

Biogenicamines

(a)

ManagedAbandoned

0000010002000300040005000600

lowast


lowast

Cellulose

Emicellulose lowast

Chitinlowast


lowast

Organicacidslowast

lowastAminoacids

lowast

Biogenicamines

(b)















4 Conclusions


Acknowledgment


References









































Journal of












Volume 2014

Advances in









Geophysics








































0000010002000300040005000600

ManagedAbandoned

lowast


Cellulose

Emicellulose

Chitinlowast


lowast

Organicacidslowast

lowast Aminoacids

lowast

Biogenicamines

(a)

ManagedAbandoned

0000010002000300040005000600

lowast


lowast

Cellulose

Emicellulose lowast

Chitinlowast


lowast

Organicacidslowast

lowastAminoacids

lowast

Biogenicamines

(b)















4 Conclusions


Acknowledgment


References









































Journal of












Volume 2014

Advances in









Geophysics






















4 Conclusions


Acknowledgment


References









































Journal of












Volume 2014

Advances in









Geophysics






































Journal of












Volume 2014

Advances in









Geophysics














Research Article Chemical, Biochemical, and Microbiological Properties...

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