Differential responses of structural and functional aspects of soil microbes and nematodes to abiotic and biotic modifications of the soil environment

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  • Applied Soil Ecology 61 (2012) 26 33

    Contents lists available at SciVerse ScienceDirect

    Applied Soil Ecology

    journa l h o me page: www.elsev ier .com

    Differe naand ne ion

    E.M. Pap okrJ. Diamana Department ob Department oc Laboratory of

    a r t i c l

    Article history:Received 27 SeReceived in reAccepted 6 Ap

    Keywords:Phospholipid fatty acidsEcoplatesNematode trophic guildsMediterraneanIrrigationManure applic

    c andunitieed PL

    structure and functional indices in plots that differed in irrigation (abiotic modication) and manureapplication (biotic modication). Four treatments were created: low water supply-manure fertilization,low water supply-no fertilization, high water supply-manure fertilization and high water supply-no fer-tilization. Two samplings were carried out: the rst after 8 weeks of irrigation (March) and the secondafter 20 weeks (June).

    The PLFA groups were mainly affected by the abiotic treatment (water supply), which acted either

    1. Introdu

    The soil changes, eitsoil componquestion rachanges of soil commuWhisenant functioningdegraded laimportant fup a eld exwater suppmodicationematode c

    CorresponE-mail add

    0929-1393/$ http://dx.doi.o soils

    ationalone in June or in combination with the biotic treatment in March. In all plots, there was an increase ofbacterial and to a lesser extent of fungal biomass from March to June, as well as a shift of the bacterialcommunity towards Gram+. The microbial catabolic prole was different in the two sampling occasions,whereas the microbial functional diversity (Shannon index) was not affected either by treatment or bysampling time. Nematode abundance was also higher in June in relation to March in all plots, whereasthe response of nematode feeding groups to treatments was occasional and related to manure ratherthan to water supply.

    2012 Elsevier B.V. All rights reserved.


    environment is subject to a plethora of abiotic and bioticher natural or induced by humans, and the response ofents to them has been studied thoroughly. However, arely addressed by soil biologists is whether the abioticthe soil environment are more important for shapingnities than the biotic ones or vice versa. According to(1999), in healthy and high resource wildlands, soil

    is mainly controlled by biotic interactions, whereas innds, modications of the abiotic environment are moreor soil functioning. Inspired by this hypothesis, we setperiment focusing on the effects of different amounts ofly (abiotic modication) and manure application (bioticn) on the structure and function of soil microbial andommunities. The application of manure is considered

    ding author. Tel.: +30 2310 998313; fax: +30 2310 998379.ress: papatheo@bio.auth.gr (E.M. Papatheodorou).

    a biotic intervention, because manure enhances microbial popula-tions and triggers bottom-up alterations of the soil community. Ourexperiment was set on a recently abandoned Mediterranean eld,which may be considered degraded, since Mediterranean soils arenutrient-poor (Parry et al., 2007), with high pH and low organicmatter (Caliskan et al., 2008), low fertility and low decomposi-tion rates (Delgado-Baquerizo et al., 2011), while they are fragile,shallow and with a distorted soil prole (Papatheodorou, 2008).Moreover, recently abandoned cultivated soils exhibit a reduced Ccontent and a deciency of N due to the lack of incorporation oforganic material into soil (McLauchlan, 2006).

    Microbial communities have attracted special interest for quan-tifying the impacts of biotic and abiotic changes of the soilenvironment, because they mediate many ecological processes insoil that are central to ecosystem functioning, such as nutrientcycling and litter decomposition, whereas they respond rapidly toenvironmental changes and stress (Winding et al., 2005). Changesin water content could impact the function and the structure oftheir communities, since dissimilar types of microorganisms aredifferentially affected by changing amounts of water potential(Todd et al., 1999; Grifths et al., 2003; Drenovsky et al., 2004).

    see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.apsoil.2012.04.002ntial responses of structural and functiomatodes to abiotic and biotic modicat

    atheodoroua,, H. Kordatosa, T. Kouserasb, N. Montopoulosa, G.P. Stamoua, M.D. Argyropouloub

    f Ecology, School of Biology, AUTH, Thessaloniki 54124, Greecef Zoology, School of Biology, AUTH, Thessaloniki 54124, Greece

    Pesticide Science, School of Agriculture, AUTH, Thessaloniki 54124, Greece

    e i n f o

    ptember 2011vised form 2 April 2012ril 2012

    a b s t r a c t

    We evaluated the effect of an abiotiand function of soil biological commdoned wheat eld. Evaluations includ/ locate /apsoi l

    l aspects of soil microbess of the soil environment

    ousosa, U. Menkissoglu-Spiroudic,

    a biotic modication of soil environment on the structures in a six month experiment established in a recently aban-FA and CLPP microbial prole as well as the nematode trophic

  • E.M. Papatheodorou et al. / Applied Soil Ecology 61 (2012) 26 33 27

    Table 1Soil physicochemical properties in the study area.

    Soil properties Mean (SE)

    Clay (%) Silt (%)Sand (%)Bulk densitypH (H2O) N-organic (%C-organic (%P (mg/100 g K (mg/g d.w

    On the othemanure is rdiversity (Cecological sing the impin numeroutodes reecin ways mfauna. Nemto vary amet al., 1999;organic mattrophic strudecompositclosely linkexhibit diffbances (Kapsoil conditio

    In ordermicrobial aregime andproles as windices. Werespond difFurthermorwe exploretreatment ishaping the

    2. Materia

    2.1. Study s

    The studNorth Greecing to 40 yKozani, the497.1 mm, rand the dry

    Soil phy(Table 1) wis classiedand alkalinand extract(MonokrouTherefore, iexpected.

    2.2. Experim

    The expwheat elding differen

    interspersed following a randomized complete design (4 treat-ments 4 replicate plots), while large untreated empty spaces wereleft between them. Once every week, eight plots were sprayed with7 L of water and the other eight with 20 L of water. These two lev-


    ere alot tplotsperiml, foue fer(highsupp

    exppliee soiwere

    of w of wfromd 20re (Ps pe.5 cm

    il ph

    ore tly c


    ucos idati


    s me



    et alws: (n chrhosp0 Sennecolumm). F37.75 (2.28)26.50 (2.64)35.75 (1.74)

    1.48 (0.035)7.87 (0.15)

    ) 0.16 (0.04)) 2.24 (0.72)d.w.) 1.7 (0.21).) 0.17 (0.01)

    r hand, several studies have shown that the addition ofesponsible for increases in soil microbial biomass andhu et al., 2007; He et al., 2008). Apart from microbes, theignicance of nematodes as bioindicators for evaluat-act of changing soil conditions has also been highlighteds studies. According to Fiscus and Neher (2002), nema-t changes in ecological structure and function of soilsore predictable and efcient than other soil ora oratode responses to soil moisture changes are reportedong trophic groups and across ecosystem types (Todd

    and references therein), while amendments involvingter inputs, such as manure application, may change thecture of their communities reecting changes in theion pathways (Tsiafouli et al., 2006, 2007). Althoughed within the soil food web, microbes and nematodeserent modes of life, different sensitivity to soil distur-agianni et al., 2010), and their responses to changingns are not synchronized (Papatheodorou et al., 2004).

    to quantify the structural and functional responses ofnd nematode communities to changing soil moisture

    manure application, we analyzed the PLFA and CLPP soilell as the nematode trophic structure and functional

    hypothesized that microbes and nematodes wouldferently to these alterations of the soil environment.e, within the context of Whisenants (1999) hypothesis,d whether in a low-resource environment the abiotics of more crucial importance than the biotic one on

    microbial and nematode communities.

    ls and methods


    y was conducted in Koila Kozanis (West Macedonia,e), between the city of Kozani and Via Egnatia. Accord-ear climatic data from the metereological station of

    mean annual temperature and rainfall is 13 C andespectively. The wet period lasts from October to May

    one from June to September.sicochemical properties of our experimental areaere determined as described in Section 2.3. The soil

    as calcaric lithosol (FAO) with a clay loam texturee pH, exhibiting low amounts of organic C, organic Nable P, compared to other Mediterranean grasslands

    els of wmean area, emeteowere stwo plthe grorain weach pof the the exOveralmanur150-F water

    Thewas apinto thplings weeksweekstaken eter anstructusampleauger 7

    2.3. So

    Befrandom(7.5 cmthe amBouyowet ox(Allen,To estias specin soil epH wa

    2.4. Ph

    Exting to Spyrouas follocolumin the pHP 589and coillary c0.25 sos et al., 2004; Papatheodorou and Stamou, 2004).t can be considered a low-resource land as originally

    ental design and sampling

    eriment was set in a recently abandoned (2 years). Sixteen experimental plots (1 m 1 m each) receiv-t treatments regarding water supply and manure were

    standard fain a GCMSture progralibrary dataimpact wasand interfaacids were solutions ocalibration supply correspond to 50% and 150% respectively of thehly precipitation of the wet period of the year in theted on the basis of 40-year climatic data of the nearbyical station. For protection from precipitation, all plotsred with plastic shield in a form of a gable roof. The

    surfaces that formed the roof triangle were touching, so that water was shed effectively and side effects ofvoided as much as possible. Channels were dug aroundo prevent surface runoff. At each irrigation level, four

    received cattle manure (4 kg/m2) at the beginning ofent, whereas in the rest four no manure was applied.

    r treatments were established: 50-F (low water supply-tilization), 50-UF (low water supply-no fertilization),

    water supply-manure fertilization), and 150-UF (highly-no fertilization).eriment lasted from January to June 2009. The manured once in the middle of January, it was incorporatedl with a mattock, and the irrigation started. Two sam-

    carried out; the rst in the middle of March (after 8atering) and the second in the middle of June (after 20atering). To study nematodes, a composite sample was

    each plot, consisting of three soil cores 3 cm in diam- cm in depth. To determine the microbial communityLFA) and its catabolic prole (Ecoplates), we took threer plot, to account for within-plot heterogeneity, by an

    in diameter and 20 cm in depth.

    ysicochemical variables

    he beginning of the experiment, ten soil samples wereollected from the experimental area by a soil auger

    cm), for the determination of soil texture, pH andts of C, N, P and K. Soil texture was estimated by the(1962) method. Soil organic C was determined by aontitration procedure using an acid dichromate system). Soil organic N was measured by the Kjeldahl method.

    extractable P, we used the method of Olsen et al. (1954), by Allen (1974). The concentration of K was determinedcts by an atomic absorption spectrophotometer. Finally,asured in a 1:5 soil/water solution.

    olipid fatty acid analysis

    n of phospholipids from soil samples was done accord-io et al. (1998) with slight modications described in. (2009). The steps of the procedure are briey describedi) extraction of lipids, (ii) separation of phospholipids byomatography, (iii) methylation of esteried fatty acidsholipid fraction, (iv) GC analysis into a Hewlett Packardries II Gas Chromatograph equipped with FID detectorted with a 5% phenyl methylsiloxane fused silica cap-n (HP-5MS 26 m length 0.320 mm i.d., lm thicknessurther conrmation of the different components of thetty acid methyl esters mixtures was achieved by analysis

    Agilent 5973 system operated in the same tempera-m and mass spectra identication based on the NIST 98

    base (Spyrou et al., 2009). Electron energy in electron 70 eV, while temperatures of the source, quadrapole,ce were 230 C, 150 C, and 280 C, respectively. Fattyquantied (nmol g1) by calibration against standardf the internal standard 19:0 ME. For this, a six-pointcurve was constructed in the range of 25200 g ml1

  • 28 E.M. Papatheodorou et al. / Applied Soil Ecology 61 (2012) 26 33

    19:0 ME. Under the above described conditions the GC response to19:0 ME was linear in the range of 25200 g ml1, with acceptablerecoveries.

    Standard nomenclature was used to describe fatty acid methylesters, basatoms: nummolecule: pof the moliso-branchiceded by a ncarboxyl en

    2.5. Carbon

    A typicaused by migroups: carstrates), am

    Soil samcollection wwere kept insuspended tion at 220 at 4 C. Fou12 ml of a swas inoculastrate utiliz24 h intervaabsorption strate absorconsidered

    2.6. Nemat

    Nematosample. Befby hand, anused the mby SJacob awool lter xed them selected ranthe genus le(1994).

    2.7. Data an

    2.7.1. PLFAThe tota

    bial biomasand conside19:0. The Pbacteria) anare consideand were chacid was ugested to band Bardgecate actinorest PLFAs and 18:1916:0, 18:0 a16:15 is kfungi (Olsso

    In orderpling on th

    components analysis (PCA) was applied to the overall PLFA data set.In order to describe differences in the composition of the microbialcommunity due to treatments a factorial ANOVA (water manure)was applied to data of the various PLFA groups from each sam-

    ccasion separately. Prior to ANOVA application, data werermed appropriately to meet the requirements of the analy-ferences among individual means were evaluated by meansBonfelity wata (

    Carbothe es ining tdiver


    D is wellrt frurthet of eing t

    nm =

    OD i coloK/2)urver ana

    the e


    et ant (cs (19

    trops anMI) fic incessgers ropo

    Enrted 0 (eguild

    2 banb, wnctie guuildes th100 indicilarl

    and or uresed on the following form: total number of carbonber of the double bonds from the methyl end of theosition of the rst double bond from the aliphatic endecule (). The prexes a and i indicate anteiso- andng, cy refers to cyclopropane fatty acids, while Me pre-umber indicate the location of methyl groups from thed of the molecule.

    utilization patterns

    l BIOLOG Ecoplate contains 31 common carbon sourcescrobial communities, which may be divided into fourbohydrates (14 substrates), carboxylic acids (9 sub-ino acids (6 substrates) and amines (2 substrates).ples were analyzed during the rst 10 days after theirith the Vahjen et al. (1995) method. Until analysis, they

    the refrigerator. Five grams from each soil sample werefor 30 min at 4 C with 10 ml of sterile 0.85% NaCl solu-rpm on a shaker. The soil slurry was precipitated for 1 hr ml of the suspension were removed and diluted withterile 0.85% NaCl solution. Each well of the Ecoplatested with 150 l of the above dilution. Bacterial sub-ation was photometrically determined at 590 nm, atls, after incubation at 28 C in the dark for 144 h. Thevalue of the control well was subtracted...


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