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Soil Biology & Biochemistry 50 (2012) 85e95

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Soil Biology & Biochemistry

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Potato cultivar type affects the structure of ammonia oxidizer communities infield soil under potato beyond the rhizosphere

Armando Cavalcante Franco Dias a, Eelco Frans Hoogwout a, Michele de Cassia Pereira e Silva a,Joana Falcão Salles a, Leonard Simon van Overbeek b, Jan Dirk van Elsas a,*

aDepartment of Microbial Ecology, Centre for Ecological and Evolutionary Studies (CEES), University of Groningen (RUG), Linneausborg, Nijenborgh 7, 9747AG, Groningen,The Netherlandsb Plant Research International, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

a r t i c l e i n f o

Article history:Received 11 October 2011Received in revised form28 February 2012Accepted 4 March 2012Available online 27 March 2012

Keywords:RhizosphereCultivarPlant growth stageSoil typeAmmonia oxidizers

* Corresponding author. Tel.: þ31 50 363 2151; faxE-mail address: j.d.van.elsas@rug.nl (J.D. van Elsas

0038-0717/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.soilbio.2012.03.006

a b s t r a c t

The effects of plants on the microbiota involved in the oxidation of ammonia in soils have beencontroversial. Here, we investigated the dynamics in the abundances and community structures of thebacterial and archaeal ammonia oxidizers (AOB and AOA, respectively) in two fields that were croppedwith potato. Six different potato cultivars were used, including a genetically-modified one, in a fourfoldreplicated experimental set-up. On the basis of bulk and rhizosphere soil extracted microbial communityDNA, AOB and AOA quantitative PCR as well as PCR-DGGE were performed. In addition, samples wereused for the production and analysis of amoA gene fragment based clone libraries. Regardless of sampletype (bulk versus rhizosphere soil) and across soils, the population sizes of AOA (of the order 104e108

amoA gene copies g�1 dry soil), were generally higher than those of AOB in the same samples (about104e105 g�1 dry soil), resulting in ratio’s of log-transformed values > 1.0. Whereas the AOB numberswere generally raised in the rhizosphere versus bulk soils in both soils, the opposite was true for the AOAnumbers. Moreover, significant effects of cultivar type on both the AOB and AOA community structureswere found in both soils, and these extended to beyond the rhizospheres. The effects were found acrossthe whole growth season. Soil type did not significantly affect the community structures of AOA, but hada small effect on the community structure of AOB. Analysis of the structures of the AOB communitiesrevealed a prevalence of AOB subgroups 2, 3a, 3b and 4 in one field soil and of 2 and 4 in the other one.With respect to the AOA, soil/sediment clusters (SS) I, II, III and IV were found to prevail.

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1. Introduction

Nitrification, i.e. the oxidation of ammonia to nitrite and subse-quently to nitrate, is an important process that governs the nitrogeneconomy of soil and is therefore crucial to plant N nutrition. In theprocess, the oxidation of ammonia to hydroxylamine and on tonitrite is the rate-limiting step (Kowalchuk and Stephen, 2001).Recently, it has become clear that ammonia oxidation is carried outnot only by ammonia oxidizing bacteria (AOB), but also by ammoniaoxidizing archaea (AOA) (Treusch et al., 2005; Leininger et al., 2006;Nicol and Schleper, 2006; Wuchter et al., 2006).

The recalcitrance of AOA and AOB to cultivation methods,possibly because of their reduced growth rates or as-yet-unknowngrowth requirements, has spurred the development of directmolecular approaches (Treusch et al., 2005; Leininger et al., 2006).

: þ31 50 363 2154.).

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Both bacterial groups can be detected by monitoring amoA, thegene that encodes the a-subunit of ammonia monooxygenase(which is involved in the first step of nitrification). Alternatively,specific regions of the 16S rRNA gene are used as markers(Kowalchuk et al., 1997; Tourna et al., 2008). Since the discovery ofthe AOA, an important focus in nitrification studies has been therespective contributions of AOA and AOB to the process (He et al.,2007; Adair and Schwartz, 2008; Chen et al., 2008; Nicol et al.,2008). Although several studies have focused on the ratiobetween AOA and AOB, we still understand little of how ecologicalfactors shape these communities and how the two groups relate toeach other (He et al., 2007; Nicol et al., 2008; Shen et al., 2008; Jiaand Conrad, 2009; Glaser et al., 2010). Factors known to affect theammonia oxidizer communities in soil are soil type (Philippot et al.,2009), pH (De Boer et al., 1995; De Boer and Kowalchuk, 2001; Nicolet al., 2008; Lehtovirta-Morley et al., 2011), temperature (Avrahamiand Conrad, 2005; Tourna et al., 2008) and ammonium concen-tration (Prosser and Nicol, 2008; Di et al., 2010; Höfferle et al.,

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e9586

2010). However, there is still much unknown about the extent towhich plants and their roots influence the AOA and AOB in soil(Kowalchuk et al., 1997; Chen et al., 2008; Philippot et al., 2009).This is in spite of our current sound knowledge on the key role ofplant roots as drivers of major soil processes by the release ofcarbonaceous and other compounds into the rhizosphere.

The increasing application of genetically-modified (GM) cropplants has raised concerns about their potential impacts on soilprocesses (Bruinsma et al., 2003). To address such effects, there isa need to first ascertain the normal effects of the crop, for instanceacross cultivars, on soil. This has been denominated the “normaloperating range” (NOR; Inceoglu et al., 2011; Pereira e Silva et al.,2011). To facilitate the definition of such a NOR, instead of assess-ing the whole complexity of the soil microbiota, indicator organ-isms (or genes) offer potentially more sensitive tools. Relevantindicators should ideally be poorly redundant, sensitive to distur-bances and easy to monitor (Bruinsma et al., 2003). Ammoniumoxidation in soil, being poorly redundant, has been proposed asa process relevant for the assessment of the normality of the soil,and hence the NOR (Bruinsma et al., 2003). Thus, both AOB and AOAmay be used as indicators, as they appear to fulfill the criteria thatdefine good indicators.

In this study, we aimed to define the AOB/AOA based NOR of soil,testing the hypothesis that potato cropping affects the abundance,diversity and structure of these ammonia oxidizing communities insoil. We therefore cross-compared these parameters across a suiteof six different potato cultivars (including a GM one) planted side-by-side in completely randomized field set-ups. To assess theeffects in different soils (Chen et al., 2008; Nicol et al., 2008; Jia andConrad, 2009), we included two soil types.

2. Materials and methods

2.1. Experimental fields, soils and potato cultivars

The experimental fields and field set-up have been describedbefore (Inceoglu et al., 2010). Briefly, two experimental fields wereused, designated Buinen and Valthermond. These fields containedsoil types representative for potato cropping in large parts of theNetherlands (described in Inceoglu et al., 2010). They were diver-gent in soil texture, organicmatter content and pH. The Buinen fieldcontained a sandy loam soil (denoted soil B), with an organicmatter content of w5% and a pH of 5.0e5.5. The Valthermond fieldcontained a sandy peat soil (denoted soil V), with an organic mattercontent of w25% and a pH of approximately 5.0. Both fields wereunder agricultural crop rotation. Prior to planting, both soilsreceived regular fertilizer, i.e. (per ha) 400 kg NP fertilizer 26e14(containing 104 kg N and 56 kg P2O5), 400 kg potassium sulfate(50%, so 200 kg K2O) and 300 kg “kalkammonsalpeter” (CaCO3mixed with NH4NO3; 27% N, so another 81 kg N).

Tubers of six different potato cultivars were seeded in each offour replicate plots per field, with the plots being randomlydistributed (completely randomized design) among four rows of sixplots each. A between-plot space of 80 cm was used. Each plotcontained 20 plants. The six potato cultivars were selected based ontheir physiological properties. This included one genetically modi-fied (GM) cultivar, denoted Modena (M) and its parental cultivarKarniko (K). Other unmodified cultivars used were Aveka (A),Aventra (Av), Désirée (D) and Premiere (P). Specifically, cultivars Aand Av grow at medium rate and their tubers had high amylopectincontent. Cultivars D and P are fast-developing cultivars, yieldingtubers with low amylopectin content. Cultivars K and M are slowly-developing cultivars. Cultivar K tubers contained high starch contentdistributed between amylose and amylopectin, whereas GM cultivarM allowed the build-up of high levels of amylopectin.

2.2. Sampling and sample analysis

From both fields, samples were taken at several time pointsduring the 2008 growth season. Rhizosphere soil samples weretaken at three time points during the season, i.e. at the young plant(EC30), flowering (EC60) and senescence stage (EC99) (Hack et al.,1993). Four individual plants were carefully removed from eachreplicate plot and loosely-adhering soil was shaken off the rootparts on-site. After this, plants were pooled per plot and taken tothe laboratory inside plastic bags. In addition, bulk soil sampleswere taken just before seeding, during the growth season at EC30,EC60 and EC99, and also in December 2008 and April 2009 (afterremoval of plants). At each sampling, one core of topsoil (12 cmdepth) was taken at each plot in the space out of reach of plantroots. During the growth season, these samples were pooled percultivar.

In the laboratory, for rhizospheres, the tightly-adhering soil wasremoved from each plant root system (four plants per replicateplot) by brushing it into the same tray. Thus, composite rhizospheresoil samples per replicate plot were produced, which were subse-quently homogenized by mixing. The bulk soil cores were directlyhomogenized by mixing.

All samples were stored in closed plastic bags (containing 1volume headspace) at room temperature for< 2 days until soil DNAextraction and analysis.

2.3. Soil pH, ammonium and nitrate contents

Soil pH was determined in 0.01 M CaCl2 (1:4.5). Nitrate N(eNO�

3 ) and ammonium N (eNHþ4 ) were determined using the

commercial kits Nanocolor Nitrat50 (detection limit, 0.3 mg N kg�1

dry weight, MachereyeNagel, Germany) and Ammonium3 (detec-tion limit, 0.04 mg N kg�1 dry weight; MachereyeNagel, Germany).Briefly, 0.2-g (rhizosphere soil) and 20-g (bulk soil) samples wereshaken head-over-head for 45 min with 4 and 20 ml of 0.01 MCaCl2, respectively. Following shaking, the extracts were filteredthrough a Millex HV Millipore filter (pore size 0.45 mm) andanalyzed according to Töwe et al. (2010).

2.4. Soil DNA extraction

Using the Powersoil DNA extraction kit (Mo Bio LaboratoriesInc., NY, USA), DNA was obtained from the samples following themanufacturer’s instructions, with the following modification. Glassbeads (0.1 mm diameter; 0.25 g) were added to the soil slurries, andbacterial cells disrupted by bead beating (mini-bead beater; Bio-Spec Products, USA) for three consecutive times (60 s). To assess thequantity and purity of the DNA, the extracts were run on 1.5%agarose gels at 90 V for 1 h in 0.5 � TAE buffer (20 mM Tris, 10 mMacetate, 0.5 mM EDTA; pH 8.0) using 5 ml of the 1-kb DNA ladder(Promega, Leiden, The Netherlands) as a molecular size andintensity marker. Gels were stained with ethidium bromide for20 min (1.2 mg ethidium bromide L�1 0.5 � TAE). The quantity ofextracted DNA was estimated using comparison to the ladder andverified using Nanodrop (ND-1000, Isogen Life Science, De Meern,The Netherlands). The DNA quality (average molecular size andpurity) was also judged from gel. Based on the gel-estimated DNAconcentrations, the samples were set (using nuclease-free water;Ambion, Nieuwerkerk a/d IJssel, The Netherlands) at a finalconcentration of 10 ng/ml.

2.5. Quantitative PCR analysis of bacterial and archaeal amoA

The abundances of AOA and AOB were determined by quanti-tative PCR (qPCR) targeting the amoA gene. For AOA, primers

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e95 87

amo23F (Tourna et al., 2008), and a degenerated version of cren-amoA616r (50-GCC ATC CAB CKR TAN GTC CA-30; Nicol et al., 2008)were used, resulting in fragments of 624 bp. AOB amoA quantifi-cation was performed using primers amoA-1F and amoA-2R (Nicolet al., 2008), generating fragments of 491 bp. The quantificationwascarried out twice from each of the four soil replicates on the ABIPrism 7300 Cycler (Applied Biosystems, Germany). The specificityof the amplification products was confirmed by melting curveanalysis, and the sizes of the amplified fragments were checked ina 1.5% agarose gel stained with ethidium bromide. Standard curveswere obtained using serial dilutions of soil-cloned archaeal orbacterial amoA genes, from 107 to 102 gene copies/ml. Possibleinhibitory effects of co-extracted humid compounds were checkedby spiking standard concentrations of target to the samples andamplifying the resulting mixes. No apparent inhibition wasobserved. All values were log-transformed. From the log targetgene numbers, we calculated the ratio AOA over AOB, where valuesof 1.0 indicate a 1:1 ratio and 2.0 a 102:1 ratio (Leininger et al.,2006).

2.6. PCR-DGGE analysis of AOA and AOB communities

The diversities and structures of the AOA and AOB communitieswere analyzed by denaturing gradient gel electrophoresis (DGGE) ofPCR-generated amplicons. As a molecular marker for the AOBcommunity, we used a region of the 16S rRNA gene, specific for theAOB of the b-proteobacterial subdivision. For amplification, a nestedPCR was used. This started with amplification of a 450-bp regionapplying forward primers CTO-A/CTO-B/CTO-C (equal amounts,final concentration 300 nM), and reverse primer CTO-R (300 nM)(Kowalchuk et al., 1997). The reaction was carried out using BioTaqpolymerase (GC Biotech, Alphen a/d Rijn, The Netherlands) using themanufacturer’s buffer. MgCl2 was added to a final concentration of2.0 mM, nucleotides at 250 nM and bovine serum albumin (BSA) at0.4 mg/ml. PCR was carried out in 25-ml volumes using the followingprogram: 93 �Ce 1min; 35 cycles of 92 �Ce 30 s, 57 �Ce 1min and68 �C e 45 s. Final extension was for 5 min at 68 �C. The resultingampliconswere diluted to 10 ng/ml, as estimated from agarose gel, toserve as templates in the second PCR reactionwith bacterial primersGC-341F and 518R (Muyzer et al., 1993). In this reaction, a 170-bpamplicon was generated, using the following program: 94 �C e

5min; 35 cycles of 94 �Ce 1min, 57 �Ce 1min, 72 �Ce 3min. Finalextension was for 30 min at 72 �C.

AOA communities were analyzed using the crenarchaeal amoAgene fragment as the molecular marker. PCR reactions were carriedout using primers CrenamoA23f and CrenamoA616r and theprotocol described by Tourna et al. (2008).

The resulting AOB amplicons were analyzed by DGGE, using anIngeny PhorU (Ingeny, Goes, The Netherlands) machine. For theanalysis, a 6% (w/v) polyacrylamide gel was prepared, containinga denaturing gradient of 35e65% (with 100% denaturant containing7 M urea and 40% formamide, deionized with AG 501-X8 Resin)(Biorad, Veenendaal, The Netherlands). After the run (16 h, 100 V,60 �C), the gels were stained using SYBR-gold (Invitrogen, Breda,The Netherlands) in 0.5 � TAE in the dark (1.5 h), after which theywere examined and photographed under UV. For the AOAcommunities, the amplicons were analyzed via DGGE usinga gradient of 15e55%, further using the same running and stainingconditions as for the AOB amplicons.

The DGGE profiles were compared using the BrayeCurtis indexof similarity, generating a matrix in which this index was deter-mined for all possible pairs of samples. The matrix was then usedto generate nonmetric multidimensional scaling (NMDS) ordina-tions to observe patterns of similarity between samples. Thesignificance of the differences between patterns was tested using

analysis of similarity (ANOSIM, 5000 permutations; Clarke andGreen, 1988). The magnitude of the test statistic R indicates thedegree of separation between groups of samples, with a value of 1indicating complete dissimilarity and 0 no difference. The anal-yses were carried out using PRIMER 6 for Windows (PRIMER-E,Plymouth, United Kingdom). Also, correlations between commu-nity structures and soil chemical parameters were tested usingthe Global BEST test in PRIMER-E software (with Spearmancoefficient; 5000 permutations), which selects the environmentalvariables that “best” explain the community patterns, by maxi-mizing a rank correlation between their respective resemblancematrices.

2.7. Construction of clone libraries of bacterial and archaeal amoAgene fragments

To examine the dynamics of AOA and AOB communities (bulksoil B), clone libraries consisting of bacterial and archaeal amoAgene fragments were generated (using primers amoA-1R andamoA-2R for bacterial and CrenamoA23f and CrenamoA616r forarchaeal PCR). This analysis thus aimed to cross-compare the bulksoil samples at young plant, flowering and senescence stages. Theamplicons were ligated into pGEM�-T easy vectors (pGEM�242-TVector System II, Promega, Madison, WI, USA), followed by intro-duction into competent Escherichia coli JM109 cells using themanufacturer’s instructions. White colonies were picked andplated on LB agar for a second check. The cloned inserts were thensequenced (AGOWA, Berlin, Germany). The AOA and AOB amoAsequences are available in GenBank under accession numbersJN977584 e JN977601 (AOA) and JQ029885 e JQ029928 (AOB).

2.8. Statistical methods for community analysis

Sequence chromatograms were trimmed and selected for high-quality sequences using the Ribosomal Database Project (RDP)pipeline. Only sequences over 400 bp and with more than 80% ofbases presenting quality>20 at Phred/Phrap analysis were selectedfor the final data set. Check-Chimera of BELLEROPHON v.3 (http://greengenes.lbl.gov) (Huber et al., 2004) was used to obtaina subset of cleaned, non-chimeric, sequences. Rarefaction analysiswas conducted using MOTHUR (Schloss et al., 2009). From this, theChaol and ACE richness estimates were deduced. Libshuff analysiswas then performed to determine the significance of differencesbetween the libraries. A P value was estimated by 10,000 randompermutations of sequences between the libraries. Similarity anal-yses were conducted based on the unweighted pair group methodwith arithmetic means (UPGMA) using the distance betweencommunities in MOTHUR (Schloss et al., 2009), which measuresthe evolutionary distances of the sequences and comparesthe evolutionary relationships among the communities acrossenvironments.

3. Results

3.1. Growth of different potato cultivars over the season

For all cultivars in soils B as well as V, the young plant stageoccurred around 30 days post-planting (dpp), i.e. at the end of June2008. However, the growth rates differed between them. Floweringstages occurred between 50 and 60 dpp for cultivars D and P (July)and between 80 and 85 dpp for A, Av, K andM (August). Later in theseason, all plants had entered the senescence stage, i.e. between110 and 115 dpp for P and D, 135 and 140 dpp for A and 145 and150 dpp for Av, K and M.

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e9588

3.2. Characteristics (ammonium, nitrate levels and pH) of soilsamples

Table 1 shows the levels of ammonium, nitrate and pH acrosstime and treatments in soils B and V. On average, ammonium levelswere 1.4 (�0.8) mg kg�1 in soil B and 2.6 (�1.2) mg kg�1 dwt in soilV. In rhizospheres of soil B, the levels were 1.4e5.5 mg kg�1 dwt,whereas in those of soil V they were 2.1e10.3 mg kg�1 dwt. Theaverage bulk soil nitrate concentrations were 12.5 mg kg�1 dwt and23.6 mg kg�1 dwt in soils B and V, respectively. In rhizospheres ofsoil V, the levels reached 87.2 mg kg�1 dwt (senescence stage)versus 27.9 mg kg�1 dwt in those of soil B (senescence stage),respectively (Table 1). Average pH values in soil B were in the range5.02e5.50 (before 5.13; young 5.44; flowering 5.50; senescence5.37; after 5.02), with fluctuations across cultivars (Table 1). In soilV, these were 4.90e5.70 (before 5.02; young 5.14; flowering 5.70;senescence 5.42; after 4.90), without a clear pattern or trend. In soilB, rhizosphere soils consistently revealed lowered pH values atflowering (range 4.35e4.83), whereas those at senescence were inthe range 5.15e6.07. In soil V, the values were 4.80e5.45 (flower-ing) versus 4.62e4.91 (senescence).

3.3. Abundances of archaeal and bacterial ammonia oxidizers equantitative PCR

In both soils, the AOA and AOB abundances were assessed acrossthe bulk and rhizosphere samples of each cultivar, by quantifyingtheir respective amoA genes (Fig. S1). Overall, considerable fluctu-ations in the target gene numbers were observed. For soil B, theAOA abundances were 6.50 � 106e7.46 � 106 g�1 dwt (bulk) soiland 4.22 � 104e6.29 � 107 g�1 dwt (rhizosphere) soil. Overall,abundances were lower in the rhizosphere than in the bulk soilacross cultivars and growth stages (significant throughout atflowering [except for Av] and at senescence for M, K and Av). Forsoil V, the AOA abundances were 6.57 � 106e8.28 � 108 g�1 dwtbulk soil and 6.10 � 106e8.11 � 108 g�1 dwt rhizosphere soil.Decreases in such abundances in the rhizosphere were noted atflowering (significant for A and Av).

Table 1Characteristics of bulk and rhizosphere soils in fields B and V (NH4þ and NO3� concent

Bulk/Rhiz stage B soil Cultivar

Aveka Aventra

Bulk flowering pH 4.93 (0.08) 4.96 (0.12)Bulk senescence pH 4.86 (0.17) 5.03 (0.09)Rhiz flowering pH 4.51(0.01) 4.58 (0.22)Rhiz senescence pH 5.45 (0.01) 5.14 (0.16)Bulk flowering NO3� 15.2 (0.56) 17.6 (0.17)Bulk senescence NO3� 11.6 (0.77) 13.1 (0.07)Rhiz flowering NO3� 21.5 (0.15) 25.7 (0.95)Rhiz senescence NO3� 24.6 (1.10) 14.1 (0.81)Bulk flowering NH4þ 1.37 (0.99) 1.09 (1.11)Bulk senescence NH4þ 2.08 (0.22) 2.99 (1.03)Rhiz flowering NH4þ 1.36 (0.06) 2.33 (0.24)Rhiz senescence NH4þ 3.07 (0.09) 2.57 (0.15)

V soila

Flowering pH 5.42 (0.51) 5.29 (0.07)Senescence pH 4.91 (0.56) 4.79 (0.03)Flowering NO3� 49.44 (0.08) 57.5 (1.91)Senescence NO3� 42.92 (1.80) 60.5 (0.62)Flowering NH4þ 4.26 (1.75) 8.91 (1.10)Senescence NH4þ 3.34 (0.76) 2.08 (0.12)

Rhiz: rhizosphere; Bulk: bulk soil.Average NO3� values before/after cropping: 9.88 (0.55) mg kg�1 and 13.7 (0.83) mg kg�

Average NH4þ values before/after cropping: 2.51 (1.04) mg kg�1 and 3.02 (0.05) mg kg�a Only bulk soil measurements (V soil).

In soil B the AOB numbers ranged from 3.35 � 103 to4.79 � 104 g�1 dwt bulk soil and from 4.72 � 104 to5.47 � 105 g�1 dwt rhizosphere soil. At all plant growth stages,these numbers were higher in the rhizosphere than in bulk soil(significant at the young plant stage throughout, and at senescencefor A). In soil V, the numbers were 3.96 � 103e4.78 � 104 g�1 dwtbulk soil and 3.87 � 103e5.41 � 105 g�1 dwt rhizosphere soil(Fig. S1). Here, the rhizosphere numbers were higher than bulk soilones at the young plant (significant for K, M and P) and floweringstages, whereas these were similar to the bulk soil ones at senes-cence stage.

We then determined the ratio’s of the AOA amoA log genenumbers over those of AOB (Fig. 1). As the AOA numbers generallyexceeded those of the AOB, the resulting ratio’s were, with oneexception, >1. In soil B, the ratio’s were in the range 1.53e1.94 forbulk and 0.82e1.29 for rhizosphere soils, revealing the ratio-depressing effect of the rhizosphere. In soil V, the range of varia-tion was 1.52e2.01 (bulk soil) and 1.16e1.91 (rhizosphere soil). Inthis soil, the ratio’s tended to decrease from young to floweringstage and then to increase from flowering to senescence stage, forthe rhizospheres of all cultivars (Fig. 1).

3.4. Structure of the AOA communities in bulk and rhizosphere soilsin two fields

Overall, the AOA PCR-DGGE patterns obtained were technicallyhighly reproducible (data not shown). Moreover, the patterns werevisually very similar across soils, cultivars, rhizosphere and time,revealing 13e18 conspicuous bands (Fig. S2). NMDS analysisrevealed clear patterns of clustering per cultivar including adjacentbulk soil (Fig. 2). This was especially true for cultivars P, D, M and A,whereas for cultivars Av and K the effect was less strong (but stillsignificant). The cultivar effects were confirmed by UPGMA-basedclusterings performed per time and soil (Figs. S3aec and S4aec).ANOSIM analysis revealed that cultivar indeed affected the AOAcommunity structures significantly, whereas soil type and soilcompartment (bulk or rhizosphere) did not exert significant effects(Table 2). Interestingly, a plant growth stage effect was observed for

rations: mg N kg�1).

Désirée Modena Karnico Premiere

4.98 (0.13) 5.06 (0.13) 4.92 (0.15) 5.02 (0.12)5.70 (0.22) 4.91 (0.18) 5.74 (0.20) 5.00 (0.07)4.35 (0.14) 4.61 (0.36) 4.83 (0.12) 4.74 (0.44)5.74 (0.07) 5.33 (0.07) 6.07 (0.07) 5.56 (0.24)14.0 (0.47) 14.0 (0.09) 12.0 (0.11) 13.7 (0.18)11.9 (0.99) 15.0 (0.06) 14.4 (0.01) 12.9 (1.00)25.3 (1.22) 28.3 (3.19) 14.7 (0.05) 27.9 (0.54)14.4 (0.96) 28.6 (0.77) 22.7 (0.70) 18.7 (1.01)1.11 (0.04) 2.13 (0.08) 3.00 (0.21) 2.77 (0.77)3.04 (0.08) 3.00 (0.09) 2.55 (0.19) 2.11 (0.03)3.93 (0.03) 2.47 (0.41) 5.52 (2.19) 3.37 (0.31)2.58 (0.20) 2.04 (0.33) 2.78 (1.18) 2.17 (0.08)

5.15 (0.03) 5.45 (0.03) 5.11 (0.56) 4.80 (0.28)4.77 (0.33) 4.85 (0.15) 4.73 (0.16) 4.62 (0.05)28.8 (0.04) 31.6 (2.88) 28.1 (0.11) 31.8 (0.75)87.2 (0.08) 49.8 (0.12) 65.5 (0.17) 64.8 (1.11)10.3 (0.44) 9.83 (0.55) 5.88 (0.98) 6.17 (0.88)4.12 (0.09) 4.77 (1.55) 3.53 (0.78) 3.26 (0.77)

1 dwt B soil.1 dwt B soil. Values: means (standard error).

Fig. 1. Ratio of log AOA/log AOB on the basis of the amoA gene abundances in the rhizosphere and bulk soil at different stages of growth of cultivars in Buinen (a) and Valthermond(b) soil. The bars indicate standard errors. Explanation: before, before seedling; y, young plant stage; f, flowering stage; sn, senescence stage; after, after removal of plants.

Fig. 2. Nonmetric multidimensional scaling (NMDS) analysis comparing the AOA (a) and AOB (b) community structures in different potato cultivars across two soils and time.Explanation of text at data points: B, Buinen; V, Valthermond; Y, young plant; F, flowering; S, senescence; b, bulk soil. Cultivars used: A, Aveka; Av, Aventra; K, Karnico; M, Modena;P, Premiere; D, Désireé.

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Table 2Results (global R and P values) of one-way ANOSIM for comparisons of the AOA andAOB community structure assessed by DGGE profiles between the different samples.

AOA AOB

Global R P value Global R P value

Growth stage 0.013 NS 0.026 <0.01Cultivar 0.855 <0.001 0.529 <0.001Soil compartment 0.008 NS 0.086 NSSoil type 0.008 NS 0.018 <0.05

Abbreviation: NS, not significant.

a b

Fig. 3. Venn diagram representation of the OTUs shared between AOA (a) and AOB (b)gene libraries at different plant growth stages (bulk soil). Total observed richness was19 and 41 OTUs at 97% similarity in AOA and AOB respectively.

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e9590

cultivars Av and K (Global R¼ 0.163, P< 0.001), but not for the othercultivars. Also, none of the soil parametersmeasured at the differentcultivars significantly influenced the AOA community structures.

Detailed UPGMA analyses performed per soil and time revealedthat, in soil B, the communities at cultivars K, D and Av on the onehand and cultivars M and P on the other, clustered together in onesupercluster at the young and flowering stages, whereas those atcultivar A clustered apart (Fig. S3aec). The clusterings relating to thesenescence stage did not follow this trend, as in this stage thecommunities of cultivars K, M, D and A grouped more closelytogether, while those of cultivars Av and P formed a separate cluster(Fig. S3aec). Similar analyses in soil V revealed that the patterns atcultivars K, D, A and Av were similar between the young and flow-ering stages, whereas those at cultivars P andMwere distinct at bothtimes (Fig. S4aec). In the senescence stages, the patterns grouped inone cluster encompassing cultivars Av, D and K, one with P and Mand one separate one, encompassing cultivar A (Fig. S4aec).

3.5. Structure of the AOB communities in bulk and rhizosphere soilsin two fields

Much like the patterns obtained for the AOA communities, therewas great technical reproducibility across the AOB patternsgenerated per treatment (data not shown), and the patterns, con-sisting of 9 (occasionally 6) to 15 conspicuous bands (Fig. S2B), wereagain similar across soil, cultivar, rhizosphere and time. NMDSshowed an overall pattern of ordination per cultivar (Fig. 2) and thisordering was also apparent in the UPGMA-based clusterings(Fig. S3def). Again, cultivar was shown to significantly affect thecommunity structures (Table 2). There were small, though signifi-cant (P < 0.05), effects of plant growth stage and soil type (Table 2;Fig. 2), whereas soil compartment did not exert a significant effect(Table 2). Finally, none of the soil parameters measured signifi-cantly influenced the AOB community structures.

Specifically, the UPGMA analysis performed per soil and timerevealed, for soil B, clusterings along cultivar type at the youngplant and flowering stages, with cultivars M and D joining in onesupercluster. Clustering of the communities at other cultivars inlarger groups varied, as, for instance, revealed in the cultivar Kclusterings across time (see Fig. S3def). Specifically, the profiles inthe rhizospheres of cultivars A, M and P appeared to shift betweenthe young plant and flowering stages, but were relatively stablebetween flowering and senescence. In the rhizospheres of cultivarsAv, K and D the communities showed no major changes betweenyoung plant and flowering stages, whereas those at senescencestage had shifted (Fig. 2; Fig. S3def). Similar analyses for soil V(Fig. S4def) showed that at the young plant stage the AOBcommunity patterns at cultivars M and D grouped together,whereas those at C K, Av, and Pwere distinct. In the flowering stage,three AOB community clusters were observed, i.e. (1) D/A, (2) K and(3) Av/M/D. Moreover, at senescence stage, the communities atcultivar D formed a cluster different from those of the other culti-vars (Fig. S4def).

3.6. Analysis of AOA and AOB clone libraries produced fromsoils B and V

To assess the AOA and AOB community structures in fields B andV (including an effect of time), clone libraries of AOA and AOB amoAgene fragments were created from bulk soil samples taken at theyoung, flowering and senescence growth stages. The cultivarssampled were A, D, P and K (soil B). From these cultivars, clonelibraries covering the young plant stage were generated from soil V.

Analysis of the libraries by UniFrac indicated that the AOBlibraries fell into two clusters, one encompassing the soil B/youngand soil B/flowering stages, and the other one joining soil V/youngwith soil B/senescence stage. Analysis of similarity (Venn diagrams;Fig. 3) of operational taxonomic units (OTUs; 97% cut-off) revealedthat 14/41 OTUs were shared across all libraries, being 2e9 typesunique for each library. The remainder was shared differentiallyacross a subset of the libraries. The AOA libraries revealed greatestsimilarity between the soil B/young and/flowering stage libraries,whereas the senescence stage library clustered separately fromthese (Fig. 3). Here, only 4/19 OTU types were shared across allthree libraries, with 2e7 of these being unique per growth stage(Fig. 3).

AOA amoA e From the clone libraries of archaeal amoA, 87, 95and 88 sequences were obtained from the soil B samples at theyoung plant, flowering and senescence stages, respectively. Shuf-fling revealed that the three libraries were different from eachother. Moreover, Libshuff analysis (10,000 randomizations) wasperformed to determine statistical differences between the groups(Table S1). The results showed that most of the groups weresignificantly different from each other (P < 0.0001), with theexception of the AOA community structures of soil B-flowering/soilB-senescence.

To assess the depth of sampling and OTU richness of thelibraries, rarefaction curves were created (97% cut-off criterion). Fornone of the libraries, the curves reached the plateau level (Fig. S5).The rarefaction, CHAO1 and ACE analyses showed an effect of timeon the AOA communities. On the basis of the first two criteria, at theyoung plant and senescence stages bulk soils had higher richnessthan at flowering stage (Table S2). A tree was constructed whichcombined the AOA clone libraries of the three growth stages pair-wise together in every possible combination. Cluster assignmentsmade based on Avrahami et al. (2002) and Koops et al. (2003)are shown in Fig. 4. The numbers of clones assigned to clusterSSI were 2 (young), 27 (flowering) and 54 (senescence), to clusterSSII: 2 (flowering) and 12 (senescence), to cluster SSIII: 1 (young), 1(flowering) and 1 (senescence) and to cluster SSIV: 64 (young), 81(flowering) and 14 (senescence).

BSENG12 – OTU5

Uncultured archaeon clone amoA GQ143165

BSENC07 – OTU10

Uncultured Archaeon clone amoA GQ143007

BSENF02 – OTU 4

GRASS 2 DQ534703

KR01 DQ534815

UAS 4050 C.17 1A B353489BSENE9 – OTU6

BSENC08 – OTU11

BYC07 – OTU13

BFLB04 – OTU15

Uncultured Crenarchaeote amoA EU885696

BSENC08 – OTU12

BFLH06 – OTU 16

Uncultured crenarchaeote amoA EF207218

ALPINE 4 DQ534700

A32 EU099950

QYA36 EF207212

BSENB06 – OTU8

BSENE12 – OTU7

57 A12 EU025151

JR 128 C6 DQ312270

DGGE AOA 1 AB353490

BSEND12 – OTU1

Nitrososphaera gargensis clone RHGCaA05c

RUD2 DQ534814

SOUTH BAY CR25 DQ278592

Uncultured archaeon amoA GQ143204

BFLB12 – OTU17

Uncultured Soil archaeon amoA FM867841

BSEND11 – OTU 14

Uncultured Archaeon clone amoA GQ142176

Alpine 3 DQ534699

Alpine 2 DQ534698

BSENH06 – OTU9

ES H1 5 DQ148774

ES H1 3 DQ148772

BYA05 – OTU3

BYA11 – OTU19

BYH02 – OTU2

QYA40 EF207216Uncultured crenarchaeote amoA FJ517356

BYH09 – OTU18

Uncultured crenarchaeote amoA FJ517353

Nitrosopumilus maritimus isolate SF AOA H10

Cenarchaeum symbiosum A clone C07D08

9994

100

100

100

6785

94

90

49

86

73

100

68

99

5199

100

100

69

99

9695

75

100

65

93

55

100

99

98100

66

98

99

0.02

Fig. 4. Phylogenetic affiliation of amoA gene sequences in bacterial clones retrieved from Buinen bulk (a) and Valthermond bulk (b) soil samples. Evolutionary relationships wereinferred using the Neighbour-Joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980). The scalebar indicates the number of base substitutions per site, and values in nodes indicate percentage of bootstrap values (in %, total of 1000 repetitions). There were a total of 583 alignednucleotide positions in the final data set of the bacterial amoA gene.

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e95 91

AOB amoA e Totals of 248, 230 and 271 sequences weregenerated from the samples taken from (bulk) soils at young,flowering and senescence stages (soil B), whereas 254 sequenceswere obtained from soil V sampled at young growth stage. All(cleaned) sequences fell within the Nitrosospira amoA clusters 2,3a, 3b and 4 in soil B and within clusters 2 and 4 in soil V, usingNitrosomonas amoA as the outgroup (Avrahami et al., 2002;Avrahami and Conrad, 2003; Glaser et al., 2010). Bootstrap valuesvaried, and those separating cluster 3a were 100/100 (Fig. 5a).

Three distinct clades, designated as related to clusters 3b, 2 and 4,were assigned (Fig. 3b). There was a rather uneven distribution ofnumbers of sequences over the different clusters in both soils. Insoil B, sequence numbers were as follows: cluster 2: 303 (young),189 (flowering), 132 (senescence); 3a: 87 (young), 15 (flowering),28 (senescence); 3b: 35 (young), 31 (flowering), 8 (senescence);4: 84 (young), 65 (flowering), 36 (senescence). In soil V, thesenumbers were as follows: cluster 2: 149; 4: 105 (young plantstage).

Nitrosospira sp. AJ2987230Nitrosospira sp. AJ298722Nitrosospira sp. X90821

BSENDESD09 – OTU4

BFLKAR3H05 – OTU9

BYAVEN97 – OTU19

Nitrosospira sp. AY123829

BSENDESA03 – OTU7

BFLDESC06 – OTU27

BSENDESG04 – OTU25

Nitrosospira sp. AJ298690

BYKARF07 – OTU13

BFLAVEE09 – OTU8

BYAVEA03 – OTU1

BYAVENB03 – OUT 15

BFLKAR3B02 – OTU11

BFLPREMH04 – OTU24

BFLDESE08 – OTU23

BFLPREME12 – OTU29

BFLDESE11 – OTU28

BYKARG06 – OTU21

BSENAVG12 – OTU22

Nitrosospira sp. AY123827Nitrosospira sp.AY189143Nitrosospira sp.AJ298697

BYAVENH01 – OTU17

BFLKAR3H11 – OTU5

BSENDESH05 – OTU26

BFLPREMF01 – OTU30

unknown proteobacterium AJ388582

BYDESB02 – OTU6

BYAVENC08 – OTU10

BYAVEN100 – OTU12

BYAVEN99 – OTU20

Nitrosospira briensis Z97858

BFLAVEF03 - OTU2

BFLAVEH03 – OTU31

Nitrosospira sp. DQ228462Nitrosospira sp. DQ228460Nitrosospira sp. EF175100

BFLAVEG10 – OTU3

Nitrosospira sp. DQ228461Nitrosospira briensis NBU76553

BFLAVEA09 – OTU16

Nitrosospira sp. AY189146Nitrosospira sp. AY189144Nitrosospira sp. AY189145

Nitrosospira sp. DQ228459Nitrosospira sp. DQ228458

Nitrosovibrio sp. DQ228466Nitrosospira sp. DQ228456

BFLKAR3B10 – OTU14

BYAVEN91 – OTU18

Nitrosospira sp. DQ228457Nitrosospira multiformis AY177933Nitrosospira multiformis DQ228454Nitrosospira sp. AJ238542Nitrosolobus multiformis X90822

Nitrosomonas cryotolerans AF272402Nitrosomonas europaea AF058692

Nitrosomonas nitrosa AF272404

87

93

65

57

67

51

91

89

51

53

9068

43

78

55

49

91

58

68

60

91

67

88

100

98

51

53

99

58

99

75

99

67

99

51

64

9999

10058

73

58

100

53

100

99100

0.05

Cluster 3a

Cluster 3b

Cluster 4

Cluster 2VBYKARE09 – OTU5

VBYDESE11 – OTU10

Nitrosospira sp. AJ298690

VBYKARC08 – OTU11

VBYPREA09 – OTU7

Nitrosospira sp. AY123829

VBYPREE04 – OTU14

VBYKARF05 – OTU4

Nitrosospira sp. AJ298723

Nitrosospira sp. AJ298722

Nitrosospira sp. X90821

VBYDESF01 – OTU1

VBYDESC11 – OTU6

VBYDESH10 – OTU8

VBYKARB11 – OTU13

VBYKARA05 – OTU12

VBYKARC02 – OTU3

VBYPRED09 – OTU9

VBYPREF06 – OTU2

Nitrosospira sp. AY189143

Nitrosospira sp. KAY123827

Nitrosospira sp. AJ298697

Nitrosomonas cryotolerans AF272402

Nitrosomonas europaea AF058692

Nitrosomonas nitrosa AF27240499

7889

70

56

72

95

8184

57

100 96

8488

85

71

52

97

65

0.01

Cluster 2

Cluster 4

a b

Fig. 5. Phylogenetic affiliation of amoA genes in archaeal clones retrieved from Buinen (B) bulk soil samples. Evolutionary relationships were inferred using the Neighbour-Joiningmethod (Saitou and Nei, 1987). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980). The scale bar indicates the number of basesubstitutions per site, and values in nodes indicate the bootstrap values (in %, total of 1000 repetitions). There were a total of 625 aligned nucleotide positions in the final data set ofthe archaeal amoA gene.

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e9592

4. Discussion

This study aimed to elucidate the effects of soil type, soilcompartment (bulk versus rhizosphere), potato cultivar type andplant growth stage on the AOA and AOB communities in soilunder potato, under the hypothesis that potato cropping affectsthe two communities. Moreover, we examined the effects ofa suite of six different potato cultivars in order to assess the

boundaries of the response variables. The resulting data mayestablish an AOA/AOB-based NOR for soil under potato. In thiscontext, the balance between ammonia oxidation performed byarchaea and bacteria has been described as being divergent indifferent environments, with the dominance of one group overthe other being dependent on the conditions reigning locally inhabitats (Beman et al., 2008; Erguder et al., 2009; Glaser et al.,2010; Dias et al., 2011).

Supplementary Table S1Libshuff analysis of the community structures of AOB and AOA at an OTU definitionlevel of 0.03 (97% threshold).

B e young B e senescence B e flowering V e young

AOAB e young e 0.0041* 0.0013* e

B e flowering 0.0016* 0.0001 e e

B e senescence 0.0072* e 0.0074* e

AOBB e young e 0.0014* 0.0003* 0.0017*B e flowering 0.0001 0.0015* e 0.0019*B e senescence 0.0001 e 0.0001 0.0002*V e young 0.0003* 0.0020* 0.0003* e

*P < 0.001.

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e95 93

First, analysis of the abundances of the AOA and AOB across theseason in both soils revealed fluctuating densities of these commu-nities, in the range of about 104e108 target gene copies g�1 dwt soil.The AOA numbers were, grossly speaking, higher than the AOB onesacross the board, with one exception, resulting in (log-transformed)AOA/AOB ratio’s > 1.0 (Fig. 1). Furthermore, opposing effects of thepotato rhizosphere were detected, being mostly negative for AOAversus positive for AOB. Taken together, this effect thus depressedthe ratio’s. However, the data fluctuated considerably, which mightindicate somewhat heterogeneous effects of soil on the respectiveammonia oxidizer abundances. Also, with the exception of a possibledifferential effect on the AOA, the carrying capacities for ammoniaoxidizers were roughly similar and showed a range (band width,potentially defining a NOR) in the two soils defined by the fluctua-tions. The AOA and AOB populations were found to be within the(broad) range observed for soils, with a slight to strong dominance ofthe AOA (Shen et al., 2008; Hallin et al., 2009; Schauss et al., 2009;Wessén et al., 2011). Thus, the AOA/AOB ratio’s observed in our studywere similar to those previously reported for top layers in agricul-tural soils (Leininger et al., 2006; Rasch et al., 2011).

An examination of the diversity and community structure dataacross our samples revealed that the structures of the AOAcommunities were quite similar across the two (bulk) soils, indi-cating that soil type did not influence the community make-ups.For the AOB, we detected a small effect of soil type (Table 2).Moreover, in none of the two soils a soil compartment (rhizosphereversus bulk soil) effect on the AOA and AOB was detected. It ispossible that such effects, whichmight come about as a result of theplant community interferingwith local ammonia concentration, pHor any other (indirect) factor, was minor compared to the over-riding effects on the ammonia oxidizers exerted by the soil.

Analysis of the AOA and AOB community patterns indicated thatshifts had occurred in subsequent plant growth stages in both soil Band V. For AOA, this was significant for cultivars Av and K. More-over, the effect was small but still significant for AOB. The finding oftemporal dynamics in both soils suggests that the AO communitiesare sensitive to influences from their local environments which areinduced by plant growth phase and are related to time-of-season.However, the communities showed different trends in soil Bversus V. In soil V, for most cultivars the AOA communities at theyoung plant and flowering stages were closely related, while thesewere different in the senescence stage. In soil B, some cultivarsshowed a difference only between young plant and flowering, someonly between flowering and senescence. Only one cultivar, P,showed a difference between each of the growth stages.

The findings of fluctuations or shifts in the relative abundancesand community structures of the AOA and AOB thus suggestedthese communities were dynamic. This may indicate successionalselection of different organisms that are best adapted todynamically-changing prevailing local soil conditions (Hallin et al.,2009; Zhang et al., 2009; Jiang et al., 2011). However, within theboundaries of the experiments, significant effects of the factorsammonium, nitrate and pH were not detected. Nevertheless,a remarkable effect of cultivar type on the AOA/AOB communitystructures was observed and this effect was consistent across timeand soil type (Fig. 2, Figs. S3 and S4). Avrahami et al. (2003) showedthat AOB community shifts in soil are often caused by local NPKlevels. Specifically, shifts were observed in NPK-fertilized soil ascompared to control soil, resulting in increased potential nitrifica-tion. Nitrogen fertilizer was recently shown to also influence theAOA community compositions (Yao et al., 2011). Taking these datatogether, the level and form of ammonium in soil may actually bea key driver of AOA/AOB communities. Thus, added ammonium, e.g.as a sulfate of chloride salt, will impact the local oxidizers, and sowill the ammonium that becomes available from the plant (or soil)

in the form of decaying organic material. The resulting ammoniumpool, a combination of both sources, may thus be the key driver ofsoil AOB/AOA communities. The clustering along cultivar found byus might relate to any ephemeral differential local, and cultivar-specific, level of ammonium (which were unfortunately notdetected by us). However, assignment of the effect to such a causehas to await more thorough assessments of the available localammonium pools.

The phylogenetic analyses based on the archaeal amoA genesconfirmed the dynamism in the communities across time.Furthermore, the sequences were found to be similar to thosefound in previous studies, suggesting that the AOA communities inthe two soils were comparable to those reported previously. Forinstance, recently Glaser et al. (2010) found most archaeal amoAsequences to be positioned in the so-called “soil cluster”. They alsoreported that AOB dominate over AOA in ammonium oxidation inbulk and rhizosphere soil at barley. Analysis of the bacterial amoAgene diversity in our study (after Avrahami and Conrad, 2003),indicated some dynamism. In concreto, dominance of Nitrosospiraclusters 2 and 4 was shown in both soil B and V (clusters 3a and 3bco-dominated in the former soil). Moreover, both clusters 2 and 4occurred at rather similar prevalences across the different cultivars.A commonality in the two soils was their relative acidity andexposure to relatively low temperature. In this respect, our datawere consistent with those of Stephen et al. (1996), who founda trend towards a dominance of Nitrosospira clusters 2 and 4 inacidic (pH 4.2) soils versus cluster 3 in other soils (agriculturalparcels). Furthermore, decreasing densities of clusters 3a and 3b inresponse to temperature drops, versus cluster 3a dominatedcommunities at high temperature have been observed (Avrahamiand Conrad, 2003; Avrahami et al., 2003). Finally, Nitrosospiraclusters 2 and 4 have so far only been found in cold soils and soappear to be typical for these (Avrahami and Conrad, 2005; Rouxet al., 2008).

Acknowledgements

We thank the NWO-ERGO programme for funding andcolleagues at Avebe (Peter Bruinenberg, Paul Heeres) for help withthe field work. EFH acknowledges the help received from theJ. Prosser group colleagues during his stay in Aberdeen. ACFD wassupported by a grant from FAPESP (2008/54013-8).

Appendix A. Supplementary data

Supplementary data related to this article can be found online atdoi:10.1016/j.soilbio.2012.03.006.

Appendix B

Supplementary Table S2Number of sequences, OTUs and richness indices for ammonia oxidizing bacteria(AOB) and archaea (AOA). Values were estimated by grouping sequences in OTUs at97% similarity.

Targeted communities Number ofsequences/OTUs

Richness indices

ChaoI ACE

AOAB e young 87/8 14 (8e45) 18 (11e42)B e flowering 95/8 9 (8e18) 11 (8e32)B e senescence 88/12 15 (12e34) 17 (13e43)

AOBB e young 271/21 26 (17e68) 20 (16e37)B e flowering 248/16 31 (22e73) 23 (21e34)B e senescence 230/9 18 (18e26) 19 (18e28)V e young 254/14 16 (14e21) 26 (24e38)

A.C.F. Dias et al. / Soil Biology & Biochemistry 50 (2012) 85e9594

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