SOIL MICROBIOLOGY

Impact of Long-Term Fertilization on the Compositionof Denitrifier Communities Based on Nitrite ReductaseAnalyses in a Paddy Soil

Zhe Chen & Xiqian Luo & Ronggui Hu & Minna Wu &

Jinshui Wu & Wenxue Wei

Received: 14 January 2010 /Accepted: 5 June 2010 /Published online: 19 June 2010# Springer Science+Business Media, LLC 2010

Abstract The effect of long-term fertilization on soil-denitrifying communities was determined by measuringthe abundance and diversity of the nitrite reductase genesnirK and nirS. Soil samples were collected from plots of along-term fertilization experiment started in 1990, locatedin Taoyuan (110°72″ E, 28°52″ N), China. The treatmentswere no fertilizer (NF), urea (UR), balanced mineralfertilizers (BM), and BM combined with rice straw(BMR). The abundance, diversity, and composition of thesoil-denitrifying bacteria were determined by using real-time quantitative PCR, terminal restriction fragment lengthpolymorphism (T-RFLP), and cloning and sequencing ofnirK and nirS genes. There was a pronounced difference inthe community composition and diversity of nirK-contain-ing denitrifiers responding to the long-term fertilizationregimes; however, less variation was observed in commu-nities of nirS-containing denitrifiers, indicating that deni-trifiers possessing nirK were more sensitive to thefertilization practices than those with nirS. In contrast,

fertilization regimes had similar effects on the copynumbers of nirK and nirS genes. The BMR treatment hadthe highest copy numbers of nirK and nirS, followed by thetwo mineral fertilization regimes (UR and BM), and thelowest was in the NF treatment. Of the measured soilparameters, the differences in the community compositionof nirK and the abundance of nir denitrifiers were highlycorrelated with the soil carbon content. Therefore, long-term fertilization resulted in a strong impact on thecommunity structure of nirK populations only, and totalorganic carbon was the dominant factor in relation to thevariations of nir community sizes.

Introduction

Denitrification, a microbially mediated process in thecycling of nitrogen in ecosystems, is a facultative respira-tory process in which oxidized nitrogen compounds areused as alternative electron acceptors for energy productionwhen oxygen is limited [1]. Denitrification has been thefocus of numerous studies because it is a major cause ofnitrogen loss from fertilized agricultural soils and contrib-utes to the production of the greenhouse gas N2O, whichaccounts for approximately 6% of the current globalwarming potential [2]. Although previous studies haverevealed that soil physical and chemical conditions indi-rectly affect denitrification [3–5], microbial factors such asthe diversity, composition, and abundance of denitrifiers areof great importance for directly regulating the N2O flux [6,7]. Therefore, it is necessary to understand the communitydynamics and abundance of soil denitrifiers as well as theirresponse to environmental changes.

As only 0.1% to 5% of the total soil bacteria communitycan be cultivated, classical culture-dependent techniques

Electronic supplementary material The online version of this article(doi:10.1007/s00248-010-9700-z) contains supplementary material,which is available to authorized users.

Z. Chen :X. Luo :M. Wu : J. Wu :W. Wei (*)Key Laboratory of Agro-ecological Processes in Subtropical Region,Institute of Subtropical Agriculture, Chinese Academy of Sciences,Changsha 410125, Chinae-mail: wenxuewei@isa.ac.cn

Z. ChenGraduate University of Chinese Academy of Sciences,Beijing 100049, China

X. Luo :R. HuCollege of Resource and Environment,Huazhong Agricultural University,Wuhan 430070, China

Microb Ecol (2010) 60:850–861DOI 10.1007/s00248-010-9700-z

are insufficient for studying the microbial communities [8].Approaches based on 16S rRNA gene sequences avoid thelimitations of cultivability and have been used to detect thebacteria community in soil samples. However, the highphylogenetic diversity among denitrifiers excludes 16SrRNA-based approaches and therefore requires the use offunctional genes which encode for enzymes directlyinvolved in the denitrification process. These include nitratereductase (narG or napA), nitrite reductases (nirK andnirS), nitric oxide reductase (cnorB or qnorB), and nitrousoxide reductase (nosZ). The reduction of nitrite to nitricoxide by nitrite reductase is the first step that distinguishesdenitrifiers from nitrate-respiring bacteria, which do notreduce nitrite to gas [9]. As a key enzyme in thedenitrification process, nitrite reductase has been used as amolecular marker for denitrifying bacteria as only denitri-fiers possess this enzyme [10]. Two structurally differentnitrite reductases are found among denitrifiers [9]: onecontains copper (Cu-nir) and is encoded by the nirK gene,and the other contains heme c and heme d1 (cd1-nir) and isencoded by the nirS gene. They also contrast in that nirKgene phylogeny is largely incongruent with 16S rRNA genephylogenies, whereas nirS gene phylogeny is mostlycongruent with the 16S rRNA gene phylogeny at thefamily or genus level [11]. The two genes seem to occurmutually exclusively in a given strain, but both types havealso been found in different strains of the same species [12].

Fertilization practices including nitrogen and organicfertilizers can stimulate both nitrification and denitrifica-tion, resulting in nitrogen loss [13–15]. Returning cropresidues to soil is highly recommended in China ascultivation is exhausting the organic matter content of soilsand a large proportion of crop residues are burned annually.The addition of crop residues to the soil provides not only asource of easily degradable organic C and N but alsoconsiderable amounts of other nutrients for plants over time[16, 17]. It is typical practice for chemical fertilizers to alsobe applied with crop residues. In mineral fertilizers,nitrogen, phosphorus, and potassium are the major elementsapplied. Urea is the main form of nitrogen fertilizer appliedin rice paddy fields and accounts for approximately 60%and 66% of the total N fertilizer used in China (22.4×106

ton N) and Asia (46.9×106 ton N), respectively [18].Although researchers have previously compared the effectsof some organic (manure and sewage sludge) and inorganicfertilizers on the composition of the denitrifying communityin upland soil [13–15, 19, 20], little is known about theeffect of fertilizers on denitrifier abundance and communitystructure in a paddy field, and no studies have beenconducted on urea and straw residues.

Rice paddy fields are distributed widely over the world,but 90% of them are located in Asia. China is one of themajor rice producers, accounting for 28% of all paddy

fields globally [18]. Denitrification is the major nitrogentransformation process in rice paddy fields due to theanaerobic conditions in soil and considerable amounts ofN2O emissions occur during the dry phase of the dry–wetcycles [21, 22]. The majority of previous studies havefocused on the influence of soil physical and chemicalconditions on denitrification, but its microbial drivingmechanisms are poorly understood.

In the present study, we used multiple moleculartechniques to investigate the influence of long-termfertilization regimes on the denitrifying community com-position, diversity, and abundance in paddy soil using nirKand nirS as genetic markers.

Materials and Methods

Soil Sampling and Processing

Soil samples were collected inMarch 2007 from the long-termfertilization experiment site (established in 1990) located inthe Taoyuan Agro-ecosystem Research Station (110°72″ E,28°52″ N), China. The soil is developed from a Quaternaryred soil, and the cropping system was double-cropped rice.Soil samples were taken from the four treatments: no fertilizer(NF), urea (UR) (182.3 kg N ha−1 a−1 as urea), balancedmineral fertilizer (BM) (182.3 kg N, 39.3 kg P, and197.2 kg K ha−1 a−1 as urea, superphosphate, and potassiumchloride, respectively), and BM combined with rice strawproduced in the plot (BMR). All the treatments have threereplicate plots which were randomly arranged in the field,with each plot having an area of 33 m2. All plots weresampled independently, and within each plot five soil coreswere taken from the upper layer (0–15 cm) and homogenizedby mixing. The sample from each plot was then separatedinto two parts, with approximately 200 g packed into a sterilebag and immersed in liquid nitrogen immediately and storedat −70°C. This soil was then freeze-dried using a freeze-drier(NEOCOOLE, Yamato) under sterile conditions and groundto a fine powder after the removal of root residues and storedat −70°C for further molecular analysis. The other portion(approximately 800 g) was air-dried and for soil physical andchemical determinations. The soil properties measured arepresented in Table 1.

Soil Microbial DNA Preparation

Total DNA was extracted according to Porteous et al. [23]with a few modifications as follows: freeze-dried andpowdered soil samples (0.5 g) were placed in a 2-mLcentrifuge tube, 0.8 mL of lysing solution (0.25 mol L−1

NaC1, 0.1 mol L−1 EDTA, and 4% SDS) was added, andthe samples were vortexed and incubated in a water bath at

Nitrite Reductase Genes in Paddy Soil 851

68°C for 15 min. Then, 0.2 mL of 5 mol L−1 guanidineisothiocyanate was added, and tubes were kept in a waterbath at 68°C for an additional 40 min with hand-shaking at10-min intervals. The sample was centrifuged at 12,000 gfor 5 min, and the supernatant was transferred to a 2-mLmicrocentrifuge tube. DNAwas precipitated by the additionof 0.125 volumes of 5 mol L−1 potassium acetate and 0.42volumes of 40% PEG8000 and kept at 4°C for 2 h beforecentrifugation at 12,000 g for 20 min. The DNA pellet wasdissolved by the addition of 0.9 mL 2×CTAB solution (2%CTAB, 1.4 mol L−1 NaCI, 0.1 mol L−1 EDTA) andincubated at 68°C for 10 min. An equal volume ofchloroform–isoamyl alcohol (24:1) was added, and themixture was centrifuged at 14,000 g for 10 min. Theaqueous phase was transferred to a new 2-mL centrifugetube and the DNAwas precipitated again by the addition of0.1 volumes of 3 mol L−1 sodium acetate and 0.6 volumesof isopropanol, and the tube was then kept at roomtemperature for 1 h. The solution was centrifuged at14,000 g for 10 min and the pellet was washed twice with0.7 mL 70% ethanol. After the pellet had dried, 0.05 mL ofsterilized H2O was added to dissolve the DNA, and thesample was stored at −20°C.

For clone library construction, the template DNA of eachtreatment was a mixture of an equal amount of DNA fromthe three replicates. For terminal restriction fragment lengthpolymorphism (T-RFLP) and real-time polymerase chainreaction (PCR) assays, triplicate DNA samples wereanalyzed independently.

Primers for nir Gene Fragments

Degenerate primers nirK-517F/1055R were modified byCunir3 and R3Cu, respectively [24, 25]. Primer set nirS-263F/950R was designed in this study. In total, 16 sequencesof nirK and 19 sequences of nirS from various strains(“Electronic supplementary material”, Table S1), derivedmostly from soil, were retrieved from the GenBank data-bases and aligned using ClustalW2 software (www.ebi.ac.uk/clustalw). Conserved peptide sequences were used to designforward and reverse degenerate primers (Table 2). Thedegeneracy of the primers was less than 100, and the target

fragment lengths of nirK and nirS were 539 and 688 bp,respectively. For T-RFLP, the forward primers were fluo-rescently labeled with 6-Fam (6-carboxyfluoresein). Inaddition, more than 30 strains were used to evaluate primerspecificity (“Electronic supplementary material”, Table S2).They were obtained from the Deutsche Sammlung vonMikroorganismen und Zellkulturen, China General Microbi-ological Culture Collection Center, Agricultural CultureCollection of China, Culture Collection Center of Guang-dong Microbial Institute, Institute of Applied Ecology, andInstitute of Subtropical Agriculture, The Chinese Academyof Sciences.

PCR Amplification and T-RFLP Determination

Fragments of nirK and nirS genes were amplified using theprimer pairs of nirK-517F/1055R and nirS-263F/950R,respectively. Each individual PCR (total volume 50 μl)contained about 60 ng of DNA template, 400 nM dNTP,400 nM of each primer, 1.5 mM Mg2+ buffer, and 2 U TaqDNA polymerase (Tiangen, China). Touchdown (TD) PCRwas performed in an Eppendorf Mastercycler (Model-5333).The program for nirK was as follows: following 5 min ofdenaturation at 95°C, 15 cycles in a touchdown program(94°C for 30 s, 57°C for 45 s, and 72°C for 60 s, followed bya 2-°C decrease of the annealing temperature every threecycles). After the completion of the TD program, 25 cycles

Table 1 Soil chemical properties

Treatment Organic carbon(g/kg)

Total N(gkg−1)

NO3−–N

(gkg−1)NH4

+–N(gkg−1)

C/N pH DEA ng N2O–Ng−1,

ds h−1

NF 18.1 (0.7) c 1.77 (0.09) c 0.16 (0.01) c 3.35 (0.52) b 10.3 (0.56) 5.17 (0.08) 68.3 (16.8) c

UR 20.1 (0.8) bc 1.91 (0.17) bc 3.16 (0.12) a 6.07 (0.68) a 10.6 (0.49) 5.17 (0.11) 220.0 (27.3) b

BM 21.6 (0.5) b 1.97 (0.14) bc 1.27 (0.02) b 3.20 (0.28) b 11.0 (0.59) 5.10 (0.09) 161.7 (31.9) b

BMR 27.4 (0.3) a 2.69 (0.06) a 1.26 (0.03) b 4.74 (0.19) ab 10.2 (0.14) 5.05 (0.22) 394.0 (23.6) a

Significant differences (p<0.05) between the treatments are shown with run-on letters: a, b, or c; mean (SEM), n=3 for the treatment

Table 2 Primer sequences, peptide templates, and positions

Primera Conserved peptide Primer sequenceb (5′–3′)

nirK517F F V Y H C A P TTYGTSTAYCACTGCGCVCC

nirK1055R N/S H N L I E A/G GCYTCGATCAGRTTRTGGTT

nirS263F L R K G A T G K TGCGYAARGGGGCNACBGGCAA

nirS950R H P E P R VA GCBACRCGSGGYTCSGGATG

a The forward and reverse primers are indicated by the letters F and R,respectively. The number of each primer indicates the nucleotide positioncorresponding to the nirK gene of Rhizobium etli CFN 42 (NC_007766.1)and the nirS gene of Azoarcus sp. EbN1 (YP_157499)b Y=C or T; S=C or G; V=A, C or G; R=A or G; B=C, G, or T; N=A, C, T, or G

852 Z. Chen et al.

were subsequently performed (94°C for 30 s, 47°C for 45 s,and 72°C s for 60 s), ending with a 7-min extension at 72°C.The program for nirS was as follows: following 5 min ofdenaturation at 95°C, samples were subjected to 21 cycles in atouchdown program (94°C for 30 s, 60°C for 45 s, and 72°Cfor 60 s, followed by a 1-°C decrease of the annealingtemperature every three cycles). After the completion of theTD program, 22 cycles were subsequently performed (94°Cfor 30 s, 53°C for 45 s, and 72°C s for 60 s), ending with a7-min extension at 72°C. The PCR products were analyzed byelectrophoresis on 1.0% agarose gels and visualized by UVTransilluminator Model M-26 (UVP, USA) after ethidiumbromide staining.

Two T-RFLP profiles for nirK were generated persample in separate reactions with the endonucleases TaqIand HaeIII (Takara Bio Inc., Japan). Similarly, two T-RFLPprofiles for nirS were produced using AluI and HaeIII.T-RFLP profiles were generated by Sangni Corporation(Shanghai, China) using an ABI Prism 3100 GeneticAnalyzer.

Cloning and Sequencing

The expected bands of nirK and nirS genes were excisedfrom agarose gels and purified using the Wizard SV Geland PCR clean-up Systems (Promega, Madison, WI, USA)following the manufacturer’s instructions. Purified PCRproducts were cloned into pGEM-T vector (Promega) andtransformed into Escherichia coli strain DH-5α, followedby “blue–white screening”. Plasmids with correct sizeinserts were screened through PCR with M13F and M13Rprimers. About 70 selected colonies in each treatment weresequenced by ABI Prism 3100 Genetic Analyzer (Invitro-gen Biotechnology Co., Guangzhou). All nir gene sequen-ces determined in the present study have been deposited inGenBank under the accession numbers FJ204477 toFJ204682.

Quantitative PCR

To minimize the influence of PCR inhibitors, extracted DNAwas further purified by agarose gel electrophoresis, bandexcision, and subsequent DNA purification using an AgaroseGel DNA Purification Kit (Takara). DNA concentration andpurity were then quantified by a Nanodrop ND-1000 UV–visspectrophotometer (NanoDrop Technologies). The primerpairs of nirK-876F and -1040R [1] were used for nirKamplification, and nirS-cd3aF and -R3cd [26] were used fornirS amplification under the same conditions described in thereferences. The real-time quantitative PCR assays wereperformed in a volume of 10 μl and the assay mixturecontained 2.5 ng of DNA template, 0.25×SYBR green(Invitrogen, US), 200 nM of dNTP, 150 nM of each primer,

3.5 mmol L−1 Mg2+ buffer, and 0.5 U Hot Start DNApolymerase (Takara). Two independent quantitative PCRassays were performed for each sample. Thermal cycling,fluorescence data collection, and data analysis were carriedout with the ABI Prism 7900 sequence detection systemaccording to the manufacturer’s instructions.

The standard curves for nirK and nirS genes werecreated using tenfold dilution series (ranging from 102 to107) of the plasmids containing the nirK and nirSfragments from the soil as described by Henry et al. [1].The efficiency of the reactions was 99% and 110% fornirK and nirS, respectively (based on the slope of thestandard curves). The R2 value for the two standard curveswas 0.995 (“Electronic supplementary material”, Fig. S1).One sharp peak was observed in the melt curves for bothnirK and nirS (“Electronic supplementary material”,Fig. S2). Appropriate negative control containing notemplate DNA gave null values. The presence of PCRinhibitors in DNA extracted from soil was examined by(1) diluting soil DNA extract and (2) mixing a knownamount of plasmid DNA to soil DNA extract beforeqPCR. Inhibition was not detected in any of these controls.Standard curves, negative controls, and environmentalDNA samples were amplified on a single 384-well plate.

Statistical Analysis

Sizes and relative abundances of terminal restrictionfragments (T-RFs) were quantified using PeakScan version1.0 software (Applied Biosystems, Inc.). Only fragmentswith a signal above 1% of the sum of all peak heights wereincluded in the analysis. The peak heights of T-RFs thatdiffered in size by ≤2 bp in an individual profile weresummed and considered as one fragment. T-RFs with asize of more than 90 bp were used for cluster analysis.Diversity indices were calculated with PC-ORD version5.0. Two methods were used to compare the denitrifiercommunities, LIBSHUFF (http://libshuff.mib.uga.edu)[27] and ordination techniques of correspondence analysis(CA) and canonical CA (CCA) [28]. Both statisticalmethods allow the determination of the likelihood thattwo communities are different or not. The clone librarieswere compared using LIBSHUFF with Bonferroni correc-tion of confidence levels; CA analysis then provided avisual map of the relationship between different microbialcommunities; CCA was used to assess the relationshipbetween microbial community profiles and environmentalvariables. CA and CCA were performed by CANOCOversion 4.53.

Sequence identity >96% was defined as an operationaltaxonomic unit (OTU). The clone sequences were alignedusing Clustal X (1.83), and a neighbor-joining tree wasproduced from the alignment (MEGA 4.0). Bootstrap analysis

Nitrite Reductase Genes in Paddy Soil 853

was used to estimate the reliability of the phylogeneticreconstruction (1,000 replicates). The aniA gene fromNeisseria gonorrhoeae (accession no. M97926) and the nirNgene (accession no. D84475) from Pseudomonas aeruginosawere used as outgroups for the phylogenetic distanceanalysis of the nirK and nirS sequences [29].

Results

Primer Validation and Amplification of nir Genes

Primer coverage and specificity were verified by amplifying acollection of denitrifiers. Primer sets nirK-517F/1055R andnirS-263F/950R could amplify a large spectrum of nir-containing strains of Proteobacteria (“Electronic supplemen-tary material”, Table S2), demonstrating that they werespecific to nirK and nirS for gram-negative bacteria.

Both nirK and nirS gene fragments were successfullyamplified from the triplicate plots of the four fertilizationtreatments (NF, UR, BM, and BMR) with the designedprimer sets of nirK and nirS. A total of 249 and 274 clonesgenerated by nirK-517F/1055R and nirS-263F/950R,respectively, were selected from the four treatments forsequencing (about 60–75 clones were selected from eachtreatment). Most of the sequences produced with nirK-517F/1055R and nirS-263F/950R showed similarity to nirKor nirS gene fragments in the NCBI databases. Of these,232 out of 249 clones for nirK were homologous to nirKgenes (93%) and 227 out of 274 clones for nirS werehomologous to nirS genes (83%). However, neither nirKnor nirS clones showed greater than 95% identity to thosefrom cultured denitrifiers. The identities of the nirKfragments ranged from 80% to 95% (average identity of90.7%) at the nucleotide level, while the identities of nirSfragments ranged from 72% to 82% (average identity of74.7%). The distant relatedness to those from culturedstrains implied that unidentified nir-containing denitrifierspredominated in the paddy soil.

Analysis of nir-Containing Denitrifier Communities

T-RFLP analysis was conducted for both nirK and nirSgenes of the treatments with three replicates. The T-RFsprofiles of nirK and nirS from triplicate samples of eachtreatment were similar, indicating that the results werereproducible and representative for the denitrifier commu-nities in the soils (“Electronic supplementary material”,Fig. S3). Therefore, the average relative abundances of theT-RFs for nirK with the restriction enzyme TaqI and nirSwith HaeIII from the different treatments were present inFig. 1. The dominant T-RFs of nirK varied under thedifferent fertilization regimes (Fig. 1a). Among them, the

relative abundance of T-RF 227 bp declined markedly inthe treatments of BM and BMR compared to the NF andUR treatments, especially in BMR, which had only 7%compared to 46% in NF. However, T-RF 266- and 209-bplevels were greater in the BM and BMR treatments.Furthermore, some less abundant fragments occurred inone or two treatments. For example, the T-RF 467 bp wasdetected only in the BM treatment. T-RF 127 bp was onlyin the NF and UR treatments, and T-RF 136 bp was only inthe BM and BMR treatments. T-RFs from in silico analysisdiffered by 0.5–3 bp from the respective experimental T-RFs. Only three T-RFs could match the T-RFs of thecorresponding nirK sequences of identified strains. Thedominant nirK T-RF 227 bp matched to Sinorhizobium(NP_435927 and ABR64876). The experimental T-RF530 bp corresponded to the T-RF 533 bp of Rhizobium(YP_473141). The T-RF 434 bp was similar to Ochrobac-trum (YP_001372908). These three strains all belonged tothe order of Rhizobiales, indicating that they might be thepredominant nirK-containing denitrifiers in the paddy soil.

Fewer variations in the relative abundance of nirS T-RFswith endonuclease HaeIII were observed among the treat-ments (Fig. 1b) as compared with nirK. Only one dominantT-RF (251 bp) was detected, which represented approxi-mately 50% of the total fragment abundance. Although theNF, BM, and BMR treatments showed a slightly higherrelative abundance of T-RFs 251, 402, and 243 bp,respectively, the differences were not significant, indicatingthat the proportion of the dominant nirS-containing groupschanged little in the four treatments. In contrast, a few lessabundant T-RFs were absent in specific treatments, e.g., T-RFs 127 and 318 bp did not occur in NF and UR, T-RF144 bp was not in NF, and T-RF 198 bp was absent in BM.T-RFs from the in silico analysis showed that the dominantT-RF 251 bp of nirS was consistent with Roseobacter(YP_681879, Rhodobacterales) and Azoarcus (YP_157499,Rhodocyclales); 94 and 180 bp corresponded to the T-RFsof 96 and 180 bp of Ralstonia (YP_585313, Burkholder-iales) and Paracoccus (YP_916270 and YP_165049,Rhodobacterales), respectively.

To determine the effect of fertilization regimes on the nirK-and nirS-defined communities, two statistical comparativeanalyses, LIBSHUFF and CA, were employed. The LIB-SHUFF results indicated that the differences of the sequencecomposition of nirK between the fertilization regimes weresignificant (P<0.05). However, no such differences wereobserved between the nirS gene libraries (Table 3). Similarresults were also observed by CA of T-RFs, where the nirKdenitrifier community compositions in the different fertiliza-tion treatments were clearly separated from each other exceptthat NF and UR were close (Fig. 2a) and nirS denitrifiercommunities were not separated as well as nirK (Fig. 2b).The nirS samples from the four treatments scattered around

854 Z. Chen et al.

the center of the ordination plot, indicating that fertilization hada weak effect on nirS denitrifier community composition.Among the measured abiotic factors, total organic carbon(TOC) and TN were significantly correlated with the nirKdenitrifier community composition (P=0.002 for both TOCand TN by Monte Carlo permutation test within CCAanalysis). Other abiotic parameters such as pH, C/N, andNH4

+–N were not significantly related to the nirK communitycomposition (data not shown).

The Effect of Fertilization on the Diversity of nir Genes

Long-term fertilization resulted in a shift in the diversity ofnirK but no such variation occurred in nirS, based on the T-RFLP results with restriction enzymes of TaqI and HaeIII fornirK and AluI and HaeIII for nirS (“Electronic supplemen-tary material”, Table S3). Plots amended with balancedmineral fertilizers and combined mineral fertilizers with rice

straw induced significantly a greater diversity of nirKcompared to no fertilization and the application of ureaalone. However, no significant differences were detectedbetween the BM and BMR, although the nirK diversityindex of BMR was higher than that of BM. Similarly, noremarkable difference was found between the NF and URtreatments. The diversity index of nirS among the fourfertilization treatments showed no significant differenceseven though plots receiving fertilizers possessed a greaternirS diversity index compared to NF plots.

The Effect of Fertilization on the Abundance of nir Genes

The copy number of nirK and nirS genes in the soil wasdetermined using real-time quantitative PCR assays (Table 4).The results were expressed in copy numbers per nanogram ofDNA and per gram of soil, both of which showed similartrends towards the fertilization regimes. In general, fertiliza-

0%

20%

40%

60%

80%

100%

NF UR BM BMR

Rel

ativ

e ab

unda

nce

of n

irK

T-R

Fs

(%)

530 467 434 266 264 250 230 227 224 209 206 187 173 169 162 158 155 136 127 123 120 115 98 96

266

227

209

0%

20%

40%

60%

80%

100%

NF UR BM BMR

Rel

ativ

e ab

unda

nce

of n

irS

T-R

Fs

(%)

57852440239034431829626025124321219818818015414413112712011310494

251

402

243

a bFigure 1 Average relativeabundances of nirK (a) T-RFswith endonuclease TaqI and nirS(b) T-RFs with HaeIII fromlong-term fertilizationtreatments. The relativeabundance of T-RFs is given asa percentage of the total peakheight. Fragment sizes withinthe graph indicate the sizes (bp)of the experimental T-RFs byT-RFLP

Fertilization practices nirK nirS

UR BM BMR UR BM BMR

NF Significant differences (ρa) 0.022* 0.002** 0.002** 0.661 0.147 0.073

UR Significant differences (ρ) / 0.024* 0.002** / 0.139 0.158

BM Significant differences (ρ) / / 0.003** / / 0.528

Table 3 LIBSHUFF analysis ofthe sequence composition ofclone libraries

a For comparison of two libraries,ρ-value represents the value ofsignificance, while single asteriskindicates ρ<0.05 and doubleasterisks indicate ρ<0.01

Nitrite Reductase Genes in Paddy Soil 855

tion increased the abundance of nirK and nirS. Compared tothe plots without fertilization (NF), the plots receiving UR,BM, and BMR showed significantly higher (P<0.05) nirScopy numbers. The highest nirS copy number occurred in theplots amended with BMR. Plots receiving mineral fertiliza-tion showed that their nirS copy numbers were intermediatebetween NF (lower) and BMR (higher) plots, and nodifference was detected between UR and BM. Variations ofnirK abundance among the four treatments were similar tothose of nirS except that there were no significant differencesbetween the NF and mineral fertilization treatments. Further-more, although the difference between the two mineralfertilization treatments was not statistically significant, thenirK copy number in the BM treatment increased over 30%in comparison with UR. The quantity of nirK was about 9–16times greater than that of nirS across the four treatments.

Phylogenetic Analysis

A nirK phylogenetic tree was constructed with 49 OTUsfrom all treatments and a selected published nirK reference

sequences. The sequences grouped into eight clusters(Fig. 3). Most of the nirK OTUs were grouped in clustersI, II, and III, accounting for 87% of the total clones. Incluster I, the clones from the BM treatment accounted for36% of the total, whereas the proportion was about 25% foreach of the other three treatments. Although clusters IV–VIIIcontained only 13% of the total clones, their distribution ineach treatment was uneven. For instance, no sequencesoriginating from NF grouped in clusters IV, VI, and VII.Also, no sequences retrieved from UR were detected inclusters V–VIII. Only two clusters (V and VII) were groupedwith the cultivated denitrifiers Bradyrhizobium sp. BTAi1and Mesorhizobium sp. BNC1, respectively.

The amplified nirS fragments and related referencesequences formed four clusters (Fig. 4). About 85% of theamplified sequences grouped in cluster Ib and had apredicted T-RF 251 bp. This cluster accounted for between83% and 88% of the nirS sequences from all fourtreatments, which is consistent with the observation thatthe relative abundance of the 251-bp T-RF fragment wasrelatively constant across treatments. Similarly, sequences

Table 4 Abundance of nirK and nirS gene copies in a paddy field soil samples determined by real-time quantitative PCR

Treatment DNA, μg g−1 soil ×103 copies ng−1 DNA ×108 copies g−1 soil bnirK/nirS

nirK nirS nirK nirS

NF 30.8 7.3 (1.6) c 0.45 (0.08) c 2.24 (0.2) bc 0.14(0.02) c 16.1

UR 38.1 10.0 (2.4) bc 1.09 (0.01) b 3.81 (0.7) b 0.42(0.04) b 9.1

BM 35.2 14.7 (3.5) ab 1.01 (0.11) b 5.17 (0.5)B 0.36(0.04) b 14.5

BMR 62.3 19.2 (3.2) a 1.62 (0.05) a 12.0 (0.8) a 1.01(0.03) a 11.9

a Significant differences (p<0.05) between the treatments are shown with run-on letters: a, b, or c; mean (SEM), n=3 for each treatment

Figure 2 CA analysis of T-RFLP profiles examining the effect ofdifferent fertilization regimes on the composition of nirK (a) and nirS(b) denitrifier communities in long-term fertilization plots. The nirK

and nirS distributions were based on the relative abundance of T-RFsafter restriction using TaqI and HaeIII enzymes, respectively

856 Z. Chen et al.

in cluster Ia and III represented similar proportions of thetotal sequences obtained in each of the fertilization treat-ments. Although Azoarcus sp., Roseobacter denitrificans,and sequences in cluster Ib and III have the identical T-RFsize of 251 bp, sequences in cluster Ib were probably close

to R. denitrificans (Rhodobacterales), whereas sequences incluster III might be similar to Azoarcus sp. (Rhodocyclales)based on their position in the phylogenetic tree (Fig. 4).Sequences in cluster Ia were also probably related toRhodobacterales because cluster Ia and Ib grouped together

Figure 3 Phylogenetic analysis of nirK sequences derived from long-term fertilization plots, with the aniA gene from N. gonorrhoeae(accession no. M97926) used as an outgroup. Bootstrap values(>50%) are indicated at the branch points. The fertilization treatmentsare shown in brackets, and the numbers before each treatment

abbreviation represent the respective number of clones. The numbersafter the treatment abbreviation indicate the respective sizes of the T-RFs after in silico analysis with the restriction enzyme TaqI. The scalebar represents a 10% estimated sequence divergence

Nitrite Reductase Genes in Paddy Soil 857

with the high bootstrap value, and one OTU (FJ204639) incluster Ia had the identical T-RF size of 180 bp withParacoccus denitrificans (Rhodobacterales). In contrast tothe above-mentioned dominant clusters, sequences in otherclusters II and IV were variably distributed in the fourtreatments, which might be due to the fewer detectedclones. For example, only one sequence, derived from theBM treatment, grouped with the known denitrifiers (Rho-docyclales) in cluster IV. Sequences in cluster II might berelated to Burkholderiales because three OTUs in thiscluster had the same T-RF of 96 bp with the Ralstoniametallidurans. Overall, the phylogenetic analyses indicatedthat the effect of long-term fertilization on the genetic

variation of nirK gene-defined communities was greaterthan that for the nirS gene.

Discussion

PCR is usually the first step in the molecular analysis ofdenitrifiers in environmental samples, and the design ofspecific PCR primers is crucial. A few pairs of establishedprimers for nirK and nirS were developed based on eithercultured strains or environmental nir sequences [1, 26, 30]but were lacking specificity to soil nir communities.Therefore, nir genes could not be amplified in some studies

Figure 4 Phylogenetic tree of nirS sequences from paddy field soilsunder different long-term fertilization, with the nirN gene from P.aeruginosa (accession no. D84475) used as an outgroup. Bootstrapvalues (>50%) are indicated at the branch points. The fertilizationtreatments are shown in brackets, and the numbers before each

treatment abbreviation represent the respective number of clones. Thenumbers after the treatment abbreviation indicate the respective sizesof the T-RFs after in silico analysis with the restriction enzyme HaeIII.The scale bar represents a 10% estimated sequence divergence

858 Z. Chen et al.

of soil samples [15, 29, 31]. In recent years, numerous nirsequences from soil and sediment have been made availablein the GenBank databases, facilitating the design of specificprimers for soil nirK and nirS communities. The newlydesigned primer sets of nirK-517F/1055R and nirS-263F/950R were validated by amplifying a collection of nirK-and nirS-containing denitrifiers representing a spectrum ofproteobacterial species, indicating that the primers were notrestricted to a narrow group. Furthermore, a wide diver-gence of nirK and nirS genes fragments were successfullyamplified and cloned from the paddy soil, confirming thespecificity of the primers to soil nir communities.

It was previously reported that the composition anddiversity of the denitrifying communities were affected bylong-term fertilization practices and that soil pH was adominant factor in regulating the microbial community inarable soils [14, 19]. However, we did not detect anyobvious change in soil pH between the fertilization regimes,probably because the urea applied constantly in this study isa physiologically neutral fertilizer and the frequent floodingmay buffer paddy soil from pH changes. Hence, soil pHmight not play a major role in the shift of nirK gene-definedcommunities caused by the fertilization. Instead, the CCAanalysis suggested that the soil organic carbon content wasa predominant factor influencing nirK denitrifiers. Thetransformation of TOC can supply labile nutrients andenergy which could stimulate the growth of soil bacteriaincluding nirK-containing denitrifiers [32].

A notable discovery was that the community composi-tion and diversity of nirK and nirS responded differentiallyto the fertilization treatments. The nirK-denitrifying com-munity composition clearly changed between fertilizationtreatments, whereas the nirS was less affected. Althoughcd1-nir (nirS) and Cu-nir (nirK) genes are functionallysimilar, they mostly belong to different bacterial strains [9].Few direct comparisons have been made of nirK and nirSdiversity in relation to fertilization in agricultural systems.Avrahami et al. [31] and Wolsing et al. [15] found that bothlong- and short-term fertilization influenced the nirK-denitrifying community composition. However, the varia-tion of nirS community responding to environmentalfactors lack constancy. In soils, nirS denitrifiers were notwell amplified due to primer specificity problems [15, 29,31, 33]. In aquatic system, nirS and nirK denitrifiersresponded differently to environmental conditions [34–36]. Our results suggested that, in paddy soil, nirK-typedenitrifiers are more sensitive to fertilization regimes thanare nirS-type denitrifiers. Recently, Chèneby and colleagues[20] also found a differential response of nitrate reductasegenes of napA and narG to fertilization and tillage system.These discoveries demonstrate that the functional commu-nities involved in denitrification respond differently tofertilization and environmental changes.

Based on the phylogenetic analyses of nirK and nirS, mostof the cloned nirK fragments clustered in a large groupincluding cluster I–IV without being identical to any culturedstrains. The nearest strains were Nitrosomonase sp.[AF339049] and Rhizobiales strains where the former wasreported as an exception of the Nitrosomonas as it wasplaced within the Rhizobiales clade [11]. Therefore, thisgroup of nirK denitrifiers might be related to Rhizobiales.However, because nirK sequences were found to besignificantly more similar to the nirK sequences from thesame habitat than to the nirK sequences retrieved from highlyrelated taxa and nirK denitrifiers were incongruent with 16SrRNA gene phylogeny [11, 29], it is not certain whethersome nirK denitrifiers from other orders besides Rhizobialesexisted in the group and further experimental confirmation isrequired. Unlike the features of nirK, nirS gene phylogeny iscongruent with the 16S rRNA gene phylogeny on the familyor genus level [11]. In accordance with the distribution of thenirS OTUs in the phylogenetic tree, the majority of theclones were related to the nirS of Rhodobacterales, with onlya small proportion of the clones similar to the nirS ofRhodocyclales and Burkholderiales. The dominance of nirSlike Rhodobacterales in paddy soil might indicate that theyplay an important role in denitrification.

There was an obvious difference of the occurrence ofOTUs among the fertilization treatments between nirK andnirS denitrifiers. The majority of nirK OTUs occurredspecifically in one of the treatments. In the contrast, most ofnirS OTUs (cluster Ib, representing 85% of all clones)occurred in all four treatments without a significantvariation. This suggests that nirK-denitrifying communitiesmight be sensitive to fertilization. Long-term fertilizationregimes resulted in obvious variations of nirK denitrifiersby selecting specific populations in the different treatments.Previous researchers have observed that nirS and nirKdenitrifiers responded differently to environmental gra-dients [37, 38], while the former was correlated with nitrateconcentration and the later was strongly correlated withdifferences in soil moisture [37]. Although nirS and nirKare functionally equivalent, nirS denitrifiers were relativelystable against fertilization because the majority of nirSOTUs were similar in all different treatments, and only asmall proportion of nirS denitrifiers were selected by aspecific treatment.

The quantification of denitrifying bacteria is of particularinterest because of the inherent difficulties in analyzing thisphylogenetically and genetically diverse functional com-munity. Real-time quantitative PCR was used to determinethe abundance of nir genes in this study. Interestingly, ricestraw incorporation (BMR) induced significantly highercopy numbers of both nirK and nirS genes. BMR might beassociated with the higher bioavailability of carbon, whichis beneficial for most denitrifiers that are heterotrophs and

Nitrite Reductase Genes in Paddy Soil 859

often C-limited [39, 40]. Similarly, Kandeler et al. [41]found that the soil organic matter was positively related toDNA yields, 16S rRNA, and the number of denitrifyinggenes (narG, nirK, and nosZ). We also found that nirK andnirS communities responded differently to mineral fertil-ization, although both UR and BM led to an increase of nirgene abundance compared to NF. The copy number of nirSmaintained in the BM treatment was the same as that in theapplication of urea alone, indicating that the growth anddevelopment of nirS denitrifiers might not be sensitive to abalanced application of nitrogen, phosphorus, and potassi-um compared to nitrogen alone. In contrast, the abundanceof nirK denitrifiers responding to BM treatment increasedby above 30% over NF although the difference was notstatistically significant. The mechanisms which influencethe two gene-defined communities in relation to mineralfertilization are unknown. It might possibly be due to thenirK-containing strains relying, to some degree, on phos-phorus and potassium supplies or root secretions, whereasnirS populations might not have this dependence.

Another interesting phenomenon observed in this studywas that the abundance of nirK was more than nine timeshigher than that of the nirS in all the fertilization regimes.Current studies indicate that only one copy of the nirK isfound in bacterial genomes, whereas three nirS copies canbe present in some species [42]. As all nitrite-reducingbacteria possess either nirK or nirS, the abundance of nirKdenitrifiers would be even higher than nirS denitrifiers inpaddy soil. By using the same primer sets, Yoshida et al.[43] detected a similar ratio of nirK/nirS in paddy soil;however, Kandeler et al. [41] reported a reversed resultwhere nirS denitrifiers were more abundant than nirKdenitrifiers in glacier foreland soil. Paddy soil seems topossess a higher ratio of nirK/nirS, but it is not yet knownhow this might be related to soil management andcultivation practices.

It is not clear what relationship exists between nircomposition and denitrifying activity. Denitrifying enzymeactivity (DEA) was determined to indicate the differencesof denitrification of the fertilization treatments in relation tonir denitrifiers in this study. Based on the relativeabundance of T-RFs in the treatments, the dominant nirKdenitrifier groups of T-RF 266 and 209 bp were positivelycorrelated and T-RF 227 bp was negatively correlated withDEA. However, no such obvious correlations were foundwith nirS denitrifiers. This might imply that, under thefertilization regimes, DEA responded to nirK gene compo-sition more closely than to nirS gene composition. Inaddition, nirK-denitrifers of T-RF 266 and 209 bp would beimportant contributors to DEA and T-RF 227 bp represent-ing nirK denitrifers that may not, although little is knownabout which group of nir denitrifiers could drive nitritereduction in paddy soils.

In conclusion, our study demonstrated that long-termfertilization induced a significant variation in nirK commu-nity composition, diversity, and abundance but had aweaker effect on nirS community structure (compositionand diversity), indicating that nirK communities were moresensitive to the fertilization practices. Although the abun-dance of nirK and nirS genes responded similarly to thefertilization, the copy number of nirK was more than ninetimes higher than that of nirS. Therefore, the nirKpopulation might be a more important component deter-mining nitrogen dynamics such as denitrification rates andN2O emissions. Among the fertilization regimes, rice strawcombined with mineral fertilizers had the strongest effecton nir communities. Although soil organic carbon wasobserved as a dominant factor determining the nirKcommunity structure, a more complete study of allcontrolling factors affecting the structure of nirK and nirSis needed for a better understanding of the ecology of nitritereducers and their functions in soil systems.

Acknowledgements This work was supported by the projects ofKZCX2-YW-BR-01 NSFC40771115 and KZCX2-YW-423.

References

1. Henry S, Baudoin E, Lopez-Gutierrez JC, Martin-Laurent F,Baumann A, Philippot L (2004) Quantification of denitrifyingbacteria in soils by nirK gene targeted real-time PCR. J MicrobiolMeth 59:327–335

2. Albritton DL, Meira Filho LG, Cubasch U (2001) Climate change2001: the scientific basis. Contributions of Working Group I to thethird assessment report of the Intergovernmental Panel on ClimateChange. IPCC, Cambridge University Press

3. Huang S, Pant HK, Lu J (2007) Effects of water regimes onnitrous oxide emission from soils. Ecol Eng 31:9–15

4. Hwang SJ, Hanaki K (2000) Effects of oxygen concentration andmoisture content of refuse on nitrification, denitrification andnitrous oxide production. Bioresour Technol 71:159–165

5. Bergsma TT, Robertson GP, Ostrom NE (2002) Influence of soilmoisture and land use history on denitrification end-products. JEnviron Qual 31:711–717

6. Conrad R (1996) Soil microorganisms as controllers of atmo-spheric trace gases (H2, CO, CH4, OCS, N2O, and NO). MicrobiolRev 60:609–640

7. Cavigelli MA, Robertson GP (2000) The functional significanceof denitrifier community composition in a terrestrial ecosystem.Ecology 81:1402–1414

8. Amann RI, Ludwig W, Schleifer KH (1995) Phylogeneticidentification and in-situ detection of individual microbial-cellswithout cultivation. Microbiol Rev 59:143–169

9. ZumftWG (1997) Cell biology and molecular basis of denitrification.Microbiol Mol Biol Rev 61:533–616

10. Braker G, Zhou JZ, Wu LY, Devol AH, Tiedje JM (2000) Nitritereductase genes (nirK and nirS) as functional markers to investigatediversity of denitrifying bacteria in Pacific northwest marinesediment communities. Appl Environ Microbiol 66:2096–2104

11. Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, DeVos P (2006) The incidence of nirS and nirK and their genetic

860 Z. Chen et al.

heterogeneity in cultivated denitrifiers. Environ Microbiol 8:2012–2021

12. Coyne MS, Arunakumari A, Averill BA, Tiedje JM (1989)Immunological identification and distribution of dissimilatoryheme cd1 and nonheme copper nitrite reductase in denitrifyingbacteria. Appl Environ Microbiol 55:2924–2931

13. Dambreville C, Hallet S, Nguyen C, Morvan T, Germon JC,Philippot L (2006) Structure and activity of the denitrifyingcommunity in a maize-cropped field fertilized with composted pigmanure or ammonium nitrate. FEMS Microbiol Ecol 56:119–131

14. Enwall K, Philippot L, Hallin S (2005) Activity and compositionof the denitrifying bacterial community respond differently tolong-term fertilization. Appl Environ Microbiol 71:8335–8343

15. Wolsing M, Prieme A (2004) Observation of high seasonalvariation in community structure of denitrifying bacteria in arablesoil receiving artificial fertilizer and cattle manure by determiningT-RFLP of nir gene fragments. FEMS Microbiol Ecol 48:261–271

16. Tirol-Padre A, Tsuchiya K, Inubushi K, Ladha JK (2005)Enhancing soil quality through residue management in a rice–wheat system in Fukuoka, Japan. Soil Sci Plant Nutr 51:849–860

17. Surekha K, Kumari APP, Reddy MN, Satyanarayana K, Cruz PCS(2003) Crop residue management to sustain soil fertility andirrigated rice yields. Nutr Cycl Agroecosyst 67:145–154

18. FAO (2003) Selected indicators of food and agriculture developmentin Asia-Pacific region (1992–2002). RAP publication, pp 23–24

19. Deiglmayr K, Philippot L, Hartwig UA, Kandeler E (2004)Structure and activity of the nitrate-reducing community in therhizosphere of Lolium perenne and Trifolium repens under long-term elevated atmospheric pCO2. FEMS Microbiol Ecol 49:445–454

20. Cheneby D, Brauman A, Rabary B, Philippot L (2009) Differen-tial responses of nitrate reducer community size, structure, andactivity to tillage systems. Appl Environ Microbiol 75:3180–3186

21. Cai ZC, Xing GX, Yan XY, Xu H, Tsuruta H, Yagi K, Minami K(1997) Methane and nitrous oxide emissions from rice paddyfields as affected by nitrogen fertilizers and water management.Plant Soil 196:7–14

22. Yan X, Du L, Shi S, Xing G (2000) Nitrous oxide emission fromwetland rice soil as affected by the application of controlled-availability fertilizers and mid-season aeration. Biol Fertil Soils32:60–66

23. Porteous LA, Armstrong JL, Seidler RJ, Watrud LS (1994) Aneffective method to extract DNA from environmental samples forpolymerase chain-reaction amplification and DNA fingerprintanalysis. Curr Microbiol 29:301–307

24. Hallin S, Lindgren PE (1999) PCR detection of genes encodingnitrile reductase in denitrifying bacteria. Appl Environ Microbiol65:1652–1657

25. Casciotti KL, Ward BB (2001) Dissimilatory nitrite reductasegenes from autotrophic ammonia-oxidizing bacteria. Appl Envi-ron Microbiol 67:2213–2221

26. Throback IN, Enwall K, Jarvis A, Hallin S (2004) ReassessingPCR primers targeting nirS, nirK and nosZ genes for communitysurveys of denitrifying bacteria with DGGE. FEMS MicrobiolEcol 49:401–417

27. Singleton DR, Furlong MA, Rathbun SL, Whitman WB (2001)Quantitative comparisons of 16S rRNA gene sequence libraries

from environmental samples. Appl Environ Microbiol 67:4374–4376

28. Bremer C, Braker G, Matthies D, Reuter A, Engels C, Conrad R(2007) Impact of plant functional group, plant species, andsampling time on the composition of nirK-type denitrifiercommunities in soil. Appl Environ Microbiol 73:6876–6884

29. Prieme A, Braker G, Tiedje JM (2002) Diversity of nitritereductase (nirK and nirS) gene fragments in forested upland andwetland soils. Appl Environ Microbiol 68:1893–1900

30. Braker G, Fesefeldt A, Witzel KP (1998) Development of PCRprimer systems for amplification of nitrite reductase genes (nirKand nirS) to detect denitrifying bacteria in environmental samples.Appl Environ Microbiol 64:3769–3775

31. Avrahami S, Conrad R, Braker G (2002) Effect of soil ammoniumconcentration on N2O release and on the community structure ofammonia oxidizers and denitrifiers. Appl Environ Microbiol68:5685–5692

32. Alden L, Demoling F, Baath E (2001) Rapid method ofdetermining factors limiting bacterial growth in soil. ApplEnviron Microbiol 67:1830–1838

33. Katsuyama C, Kondo N, Suwa Y, Yamagishi T, Itoh M, Ohte N,Kimura H, Nagaosa K, Kato K (2008) Denitrification activity andrelevant bacteria revealed by nitrite reductase gene fragments insoil of temperate mixed forest. Microbes Environ 23:337–345

34. Yan TF, Fields MW, Wu LY, Zu YG, Tiedje JM, Zhou JZ (2003)Molecular diversity and characterization of nitrite reductase genefragments (nirK and nirS) from nitrate- and uranium-contaminatedgroundwater. Environ Microbiol 5:13–24

35. Junier P, Kim OS, Witzel KP, Imhoff JF, Hadas O (2008) Habitatpartitioning of denitrifying bacterial communities carrying nirS ornirK genes in the stratified water column of Lake Kinneret, Israel.Aquat Microb Ecol 51:129–140

36. Falk S, Hannig M, Gliesche C, Wardenga R, Koster M, Jurgens K,Braker G (2007) nirS-containing denitrifier communities in the watercolumn and sediment of the Baltic Sea. Biogeosciences 4:255–268

37. Jones CM, Hallin S (2010) Ecological and evolutionary factorsunderlying global and local assembly of denitrifier communities.ISME J 4:633–641

38. Smith JM, Ogram A (2008) Genetic and functional variation indenitrifier populations along a short-term restoration chronose-quence. Appl Environ Microbiol 74:5615–5620

39. Cao YP, Green PG, Holden PA (2008) Microbial communitycomposition and denitrifying enzyme activities in salt marshsediments. Appl Environ Microbiol 74:7585–7595

40. Parkin TB (1987) Soil microsites as a source of denitrificationvariability. Soil Sci Soc Am J 51:1194–1199

41. Kandeler E, Deiglmayr K, Tscherko D, Bru D, Philippot L (2006)Abundance of narG, nirS, nirK, and nosZ genes of denitrifyingbacteria during primary successions of a glacier foreland. ApplEnviron Microbiol 72:5957–5962

42. Jones CM, Stres B, Rosenquist M, Hallin S (2008) Phylogeneticanalysis of nitrite, nitric oxide, and nitrous oxide respiratoryenzymes reveal a complex evolutionary history for denitrification.Mol Biol Evol 25:1955–1966

43. Yoshida M, Ishii S, Otsuka S, Senoo K (2009) Temporal shifts indiversity and quantity of nirS and nirK in a rice paddy field soil.Soil Biol Biochem 41:2044–2051

Nitrite Reductase Genes in Paddy Soil 861