Dn

JS

a

ARRA

KPnRPC

1

rlgbeSi(fiAD(e12ere

0d

Microbiological Research 167 (2012) 157– 165

Contents lists available at ScienceDirect

Microbiological Research

jou rn al h omepage: www.elsev ier .de /micres

iversity of free-living nitrogen-fixing microorganisms in the rhizosphere andon-rhizosphere of pioneer plants growing on wastelands of copper mine tailings

ing Zhan, Qingye Sun ∗

chool of Resources and Environmental Engineering, Anhui University, Hefei, Anhui 230601, PR China

r t i c l e i n f o

rticle history:eceived 2 December 2010eceived in revised form 15 May 2011ccepted 22 May 2011

eywords:CR-DGGEifH genehizosphereioneer plants

a b s t r a c t

The composition of free-living nitrogen-fixing microbial communities in rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings was studied by the presenceof nifH genes using Polymerase Chain Reaction-Denatured Gradient Gel Electrophoresis (PCR-DGGE)approach. Eleven rhizosphere tailing samples and nine non-rhizosphere tailing samples from six plantcommunities were collected from two wastelands with different discarded periods. The nested PCRmethod was used to amplify the nifH genes from environmental DNA extracted from tailing sam-ples. Twenty-two of 37 nifH gene sequences retrieved from DGGE gels clustered in Proteobacteria(�-Proteobacteria and �-Proteobacteria) and 15 nifH gene sequences in Cyanobacteria. Most nifH genefragments sequenced were closely related to uncultured bacteria and cyanobacteria and exhibited less

opper mine tailings than 90% nucleotide acid identity with bacteria in the database, suggesting that the nifH gene fragmentsdetected in copper mine tailings may represent novel sequences of nitrogen-fixers. Our results indicatedthat the non-rhizosphere tailings generally presented higher diversity of nitrogen-fixers than rhizospheretailings and the diversity of free-living nitrogen-fixers in tailing samples was mainly affected by thephysico-chemical properties of the wastelands and plant species, especially the changes of nutrient and

sed b

heavy metal contents cau

. Introduction

The majority of nitrogen fixation in terrestrial ecosystems is car-ied out by symbiotic bacteria in association with plants (especiallyeguminous plants), however, free-living nitrogen-fixing microor-anisms inhabiting soils can significantly contribute to the Nudgets of a number of ecosystems (Kahindi et al. 1997; Deslippet al. 2005; Unkovich and Baldock 2008; Hsu and Buckley 2009).ome nitrogen-fixers can promote plant growth by synthesiz-ng and releasing antibiotics and growth-promoting substancesAquilanti et al. 2004; Beneduzi et al. 2008). Free-living nitrogen-xers of soil or on soil surface include Cyanobacteria, Proteobacteria,rchaea and Firmicutes (Kahindi et al. 1997; Widmer et al. 1999;iallo et al. 2004; Duc et al. 2009). Nutrient content of substrate

Widmer et al. 1999; Tan et al. 2003; Zhang et al. 2006; Coelhot al. 2008, 2009; Duc et al. 2009), soil pollution (McGrath et al.995; Mårtensson and Torsrensson 1996; Oliveira and Pampulha006; Oliveira et al. 2009), plant species (Tan et al. 2003; Diallo

t al. 2004; Duc et al. 2009), plant genotype (Tan et al. 2003), planthizosphere (Hütsch et al. 2002; Soares et al. 2006; Wartiainent al. 2008; Coelho et al. 2009; Duc et al. 2009; Sato et al. 2009),

∗ Corresponding author. Tel.: +86 551 3861882; fax: +86 551 3861882.E-mail addresses: sunqingye@ahu.edu.cn, zhanjmyx@sina.com (Q. Sun).

944-5013/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.micres.2011.05.006

y the colonization of plant community.© 2011 Elsevier GmbH. All rights reserved.

temperature (Deslippe et al. 2005), etc., can affect nitrogenaseactivity and composition of free-living nitrogen-fixing microbialcommunities of soil. The nifH gene, encoding the iron protein sub-unit of nitrogenase, is highly conserved among diazotrophs whichprovides evidence for potential nitrogen fixation (Coelho et al.2009). In recent years, the nifH gene in environmental samples hasbeen widely studied using molecular methods to investigate thestructure of diazotrophic community under various environmen-tal conditions (Widmer et al. 1999; Diallo et al. 2004; Deslippe et al.2005; Soares et al. 2006; Zhang et al. 2006; Coelho et al. 2008, 2009;Wartiainen et al. 2008).

The wastelands of copper mine tailings produced by the floc-culate flotation of copper ore contain very low nutrient levelsand only some tolerant plant species can naturally colonize thesewastelands (Sun et al. 2004). Most tolerant plants growing onwastelands of copper mine tailings in Tongling, East China, belongto Gramineae and Compositae without symbiotic nitrogen fix-ation (Sun et al. 2004; Tian et al. 2005), such as Phragmitesaustralis, Imperata cylindrica var. major, Cynodon dactylon, Mis-canthus sinensis, Miscanthus floridulus, and Conyza Canadensis. Itis reasonable to hypothesize that under the condition of these

wastelands of copper mine tailings, the nitrogen needed by pio-neer plants should mainly due to the activity of free-livingnitrogen-fixers, especially those inhabiting the rhizosphere of theseplants.

1 ical Re

tt(2mmnpfa

nnwicwct

2

2

staaMfmw

dmvcwtaaw

2

zttlap1(mst

wdIsc

58 J. Zhan, Q. Sun / Microbiolog

The rhizospheric nitrogen-fixers of pioneer plants growing onhe wastelands of copper mine tailings are poorly known whereashose of cropped and desert soils have been extensively studiedCoelho et al. 2008, 2009; Wartiainen et al. 2008; Chowdhury et al.009; Sato et al. 2009). Since the condition of these poor copperine tailings are particular variable and often with low organicatter content and with limited amounts of bioavailable inorganic

utrients, the free-living nitrogen-fixers living in association withioneer plants should possess adaptive mechanisms to cope withrequent droughts, starvation, high osmolarity, high temperaturend high erosion.

In this study, we have monitored the diversity of free-livingitrogen-fixers in rhizosphere and non-rhizosphere tailings of pio-eer plants by PCR-DGGE method. The objectives of the researchere (1) to compare the diversity of free-living nitrogen-fixers

n rhizosphere and non-rhizosphere tailings from various plantommunities with different development period in two tailingastelands; and (2) to explore the main factors resulting in the

hanges of free-living nitrogen-fixing microbial community struc-ure in wastelands of copper mine tailings.

. Materials and methods

.1. Study area

The two wastelands of copper mine tailings were the Yang-hanchong and old Tongguanshan wastelands (6.08 km betweenwo wastelands), and they are located in the Tongling Copper Minerea (30◦54′N, 117◦53′E), Anhui Province, East China. The averagennual rainfall in this area is 1346 mm, and the rainy season is fromay to September. The average annual temperature is 16.2 ◦C. The

rost-free period is of 237–258 days (Sun et al. 2004). The copperine tailings were a sandy loam and easily eroded by water andind.

The Yangshanchong and old Tongguanshan wastelands wereiscarded in 1991 and 1980, respectively. Main natural plant com-unity in the old Tongguanshan mine wasteland is I. cylindrica

ar. major community, and M. sinensis community and C. dactylonommunity are in little patches in wasteland. In Yangshanchongasteland, M. floridulus communities come forth near the edge of

he tailings dam, around which I. cylindrica var. major communitynd C. dactylon community are present; Hippochaete ramosissimumnd Zoysia sinica community distribute in low-lying areas of theasteland.

.2. Collection of samples

Sample collection was conducted in June 2008. We collected rhi-osphere samples of each pioneer plant by pulling the plant out ofhe ground and mechanically separating the soil strongly adheringo roots; non-rhizosphere tailings were collected from the upperayer under plant community (0–10 cm in depth) from the samerea. The wastelands from nine spots (at least 10 m apart) of eachlant community were pooled to be one sample, and a total of1 rhizosphere samples (∼R) and nine non-rhizosphere samples∼B) were collected for the analysis of free-living nitrogen-fixing

icrobial diversity (Table 1). All the tailings were transferred toeparate sterile screw-capped plastic tubes and transported in iceo the laboratory.

The tailing samples for physico-chemical properties analysisere simultaneously collected from areas covered by I. cylin-

rica var. major community in old Tongguanshan wasteland, and. cylindrica var. major community, M. floridulus community, Z.inica community, H. ramosissimum community and C. dactylonommunity in Yangshanchong wasteland. Three replicate samples

search 167 (2012) 157– 165

from each field were used to analyze physico-chemical propertiesand each replicate subsample was collected from the upper layer(0–10 cm in depth) of tailings under each type of plant communityin three spots.

2.3. Measurement of physical and chemical properties

The pH and electric conductivity (EC) were mea-sured using pH meter and electric conductivity method(Samplew:WaterV = 1 g:5 ml), respectively. The total nitrogen(TN) was determined by Kjeldahl method. The loss of ignition (LOI)was measured in muffle furnace at 550 ± 5 ◦C for 6 h to representtotal organic matter considering the sulfides in mine wastelands.The available phosphorus (AP) of samples was extracted using0.5 mol l−1 NaHCO3, and then determined by molybdate bluespectrophotometric method (Nanjing Institute of Soil Science,Chinese Academy of Sciences, 1978). The total metals (Pb, Zn, Cu,As, Cr and Cd) in wastelands samples were measured by inductivecoupled plasma-atomic emission spectroscopy (ICP-AES) (XSPIntrepid II, USA) after the samples were digested with a mixtureof hydrofluoric (40.0%), nitric (68.0%), and perchloric acid (72.0%).Diethylenetriamine pentaacetic acid (DTPA)-extractable metals(Pb, Zn, Cu, As, Cr and Cd) were obtained by using a mixture of5 mM DTPA, 10 mM CaCl2 and 100 mM triethanolamine (TEA) atpH 7.3 (Samplew:WaterV = 1 g:2 ml) for 2 h at 25 ◦C.

2.4. Molecular analysis

2.4.1. Nucleic acid extractionTotal DNA was extracted by SDS-based DNA extraction method

(Zhou et al. 1996). Samples (1 g) were mixed with 540 �l of DNAextraction buffer (100 mmol l−1 Tris–HCl at pH 8.0, 100 mmol l−1

sodium EDTA at pH 8.0, 100 mmol l−1 sodium phosphate at pH 8.0,1.5 mol l−1 NaCl, 1% CTAB) and 10 �l of proteinase K (10 mg ml−1)in sterilized centrifuge tubes by horizontal shaking at 225 rpmfor 30 min at 37 ◦C. Then, 60 �l of 20% SDS were added, and thesamples were incubated at 65 ◦C for 2 h with gentle end-over-endinversions every 15–20 min. The supernatants were collected aftercentrifugation at 6000 × g for 10 min at room temperature andtransferred into new sterilized centrifuge tubes. The soil pelletswere extracted two more times by adding 180 �l of the extrac-tion buffer and 20 �l of 20% SDS, vortexing for 10 s, incubating at65 ◦C for 10 min, and centrifuging as before. Supernatants fromthe three cycles of extractions were combined and mixed withan equal volume of phenol:chloroform:isoamyl alcohol (25:24:1,v/v/v), and the aqueous phase containing nucleic acids were sep-arated by centrifugation at 6000 × g for 10 min. Then, an equalvolume of chloroform:isoamyl alcohol (24:1, v/v) was added. Theaqueous phase was recovered by centrifugation (6000 × g) and pre-cipitated with 0.6 volume of isopropanol at room temperature for1 h. The pellet was obtained by centrifugation at 16,000 × g for20 min at room temperature, washed with cold 70% ethanol, andresuspended in TE (10 mmol l−1 Tris base and 1 mmol l−1 EDTA, pH8.0), to give a final volume of 50 �l. The integrity of extracted DNAwas evaluated on 0.8% agarose gel, and the concentration and purityof the extracts were estimated by spectrophotometry (NanoDrop,USA) to determine the content of DNA used for PCR amplification.

2.4.2. PCR amplification of nifH geneDNA was amplified using a nested PCR as described by Diallo

et al. (2004). The first PCR was carried out with the forward primerFGPH19 and the reverse primer PolR (Table 2; Simonet et al. 1991;

Poly et al. 2001a). The second PCR was carried out with the for-ward primer PolF containing a GC clamp and the reverse primerAQER (Table 2; Poly et al. 2001a). The 50 �l reaction mix con-tained: 2 �l of a 1:50 dilution of extracted DNA (50–100 ng), 5 �l

J. Zhan, Q. Sun / Microbiological Research 167 (2012) 157– 165 159

Table 1Samples used for microbial diversity analysis and abbreviation in the study (in bold).

Wasteland of copper mine tailings studied Types of plant communities Samples and abbreviation in paper

Old Tongguanshan I. cylindrica var. major community Rhizosphere tailings of I. cylindrica (TIR)Rhizosphere tailings of C. dactylon (TCR)Rhizosphere tailings of M. sinensis (TMR)Non-rhizosphere tailings of I. cylindrica (TIB)

Yangshanchong C. dactylon community Rhizosphere tailings of C. dactylon (YCR)Non-rhizosphere tailings of C. dactylon (YCB)

M. floridulus community Rhizosphere tailings of M. floridulus (YMR)Non-rhizosphere tailings of M. floridulus (YMB)

I. cylindrica var. major community Rhizosphere tailings of I. cylindrica in young phase (YISR)Non-rhizosphere tailings of I. cylindrica in young phase (YISB)Rhizosphere tailings of I. cylindrica in mature phase (YIMR)Non-rhizosphere tailings of I. cylindrica in mature phase (YIMB)

H. ramosissimum community Rhizosphere tailings of H. ramosissimum in young phase (YHSR)Non-rhizosphere tailings of H. ramosissimum in young phase(YHSB)Rhizosphere tailings of H. ramosissimum in mature phase (YHMR)Non-rhizosphere tailings of H. ramosissimum in mature phase(YHMB)

Z. sinica community Rhizosphere tailings of Z. sinica in young phase (YZSR)

Table 2Primers used for PCR amplification.

Primers Sequences (5′–3′)b Reference

FGPH19 TAC GGC AAR GGT GGN ATH G Simonet et al. (1991)PolR ATS GCC ATC ATY TCR CCG GA Poly et al. (2001a)AQER GAC GAT GTA GAT YTC CTG Poly et al. (2001a)PolF-GCa TGC GAY CCS AAR GCB GAC TC Poly et al. (2001a)

oomaffpf

Pf

2

pdue6GGe(ue

2

Ttmc

a GC clamp: CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC C.b R = A/G; N = A/G/C/T; H = T/C/A; Y = C/T; S = G/C; B = G/T/C.

f 10 × PCR buffer (with Mg2+) (Transgen, Beijing, China), 20 pmolf each primer, 10 �mol of each dNTPs and 2.5 U of Taq DNA poly-erase. For the second PCR, 1 �l of the first PCR product was used

s the template, with the following amplification conditions: 94 ◦Cor 3 min, 30 cycles of denaturation at 94 ◦C for 1 min, annealingor 1 min at 55 ◦C for the first PCR and at 48 ◦C for the second PCR,rimer extension at 72 ◦C for 2 min, with a final extension at 72 ◦Cor 5 min.

Negative controls (without DNA) were run in all amplifications.CR products were analyzed by 1.5% agarose gel electrophoresisollowed by staining with SYBR Green I.

.4.3. DGGE analysisThe PCR products (20–30 �l) were loaded onto 8% (w/v)

olyacrylamide–bisacrylamide (37.5:1) (Amresco, USA) gels withenaturation gradients from 45% to 70% where 100% is 7 mol l−1

rea and 40% (v/v) deionized formamide (Amresco, USA) in 1 × TAElectrophoresis buffer. Electrophoresis was carried out at 100 V at0 ◦C for 17 h (Bio-Rad, USA). Gels were then stained with SYBRreen I in l × TAE for 20 min at room temperature and observed byel Image System. Bands of interest were excised, and DNA wasluted with 30 �l Tris–EDTA buffer for 24 h. The resulting solution5 �l) was used for PCR amplification with primers PolF and AQERsed for nitrogen-fixers. The purity and correct running position ofach fragment were confirmed by further DGGE analysis.

.4.4. Cloning, sequencing and phylogenetic analysisPurified PCR products from DGGE bands were cloned into PEASY

1 Cloning vector by a rapid ligation kit according to the instruc-ions of the manufacturer (Transgen, Beijing, China). Ligation

ixtures were transformed into Trans1-T1 Phage Resistant chemi-ally competent Escherichia coli cells (Transgen, Beijing, China). The

Non-rhizosphere tailings of Z. sinica in young phase (YZSB)Rhizosphere tailings of Z. sinica in mature phase (YZMR)Non-rhizosphere tailings of Z. sinica in mature phase (YZMB)

transformed cells were plated onto Luria–Bertani agar plates in thepresence of ampicillin. After 14 h incubating at 37 ◦C, white cloneswere obtained, followed by the sequencing of single clones.

The nucleotide sequences were compared with those in theGenBank using BLAST on the NCBI’s homepage. According to thesimilarities in the BLAST hits and alignments from all the sequencesobtained, the aligned sequences (the closest relative and the clos-est one of known genus in nucleic acid database corresponding tothe DGGE bands) were used to construct a phylogenetic tree bythe neighbor-joining method with the MEGA package version 4.0(Tamura et al. 2007). The topology of this distance tree was testedby resampling data with 5000 bootstraps to provide confidenceestimates for tree topologies.

2.5. Data analyses

Digitized images of DGGE fingerprint were used to quantifydiversity as enabled by Tanon Image System which detects bandsand quantifies relative concentrations of DNA from cumulativepixel intensities within a given lane. The Shannon diversity indexwas calculated from the number of bands present and relativeintensities of bands in each lane (Diallo et al. 2004). Princi-pal components analysis (PCA) and cluster analysis were carriedout by CANOCO 4.5 and MVSP V3.1, respectively, according tothe presence and absence of bands occurred in DGGE finger-print profiles. The presence or absence of a nucleic acid bandat the same height in each lane was marked with a 1 or 0,respectively. The similarities between the DGGE patterns were dis-played graphically as a dendrogram based on UPGMA algorithms(unweighted pair group method with arithmetic averages). More-over, the physico-chemical parameters of tailing samples fromdifferent plant communities were also selected as variables for thecluster analysis (UPGMA) to determine the similarities betweensamples.

2.6. Nucleotide sequence accession numbers

The nucleotide sequences of 37 DGGE bands have beendeposited in the Genbank Data Library under accession numbersHM565827–HM565863.

1 ical Research 167 (2012) 157– 165

3

3

ispm

ma7lT1wl

3

scrtfworbgsdwmsH

cwh

Fig. 1. DGGE profiles of nifH gene from rhizosphere and non-rhizosphere tailings.

60 J. Zhan, Q. Sun / Microbiolog

. Results

.1. General wasteland situation

The properties of tailing samples from pioneer plant communityn two copper mine wastelands were shown in Table 3. It can beeen that the Tongguanshan wasteland with long discarded periodresented lower pH, higher contents of nitrogen (TN) and organicatter (LOI) than Yangshanchong wasteland.In this study, Cu and Zn were the main heavy metals in two

ine wastelands investigated, according to both the total andvailable contents. The contents of total Cu and Zn ranged in40–1670 mg kg−1 and 317–741 mg kg−1 in Yangshanchong waste-

and and Tongguanshan wasteland, respectively (data not shown).he contents of DTPA-extracted available Cu and Zn ranged from5.36 to 126.09 mg kg−1 and 9.33 to 24.40 mg kg−1 in two mineastelands, respectively. The Tongguanshan wasteland showed

ess heavy metal toxicity than Yangshanchong wasteland.

.2. Changes of DGGE profiles and diversity indices

All DNA extracted from rhizosphere and non-rhizosphere tailingamples were successfully amplified using the nested PCR proto-ol. The resulting ≈320-bp-long nifH gene fragments were thenesolved by DGGE, whose profiles showed various banding pat-erns in the different tailing samples (Fig. 1). Diversity index ofree-living nitrogen-fixers in wastelands of copper mine tailingsas assessed by determining the number and relative intensities

f DGGE bands. Comparing the rhizosphere samples with non-hizosphere samples (Fig. 2) in Yangshanchong wasteland, it cane seen that the non-rhizosphere samples (Y-B, H′ = 2.197–3.091)enerally presented higher diversity indices than the rhizosphereamples (Y-R, H′ = 1.792–2.565). In Tongguanshan wasteland, theiversity index of non-rhizosphere tailing sample (TIB, H′ = 2.890)as higher than that of rhizosphere sample from I. cylindrica var.ajor (TIR, H′ = 2.565), but was lower than that of rhizosphere

amples from C. dactylon and M. sinensis (TCR, H′ = 3.178; TMR,′ = 3.091).

Both rhizosphere and non-rhizosphere tailing samples from I.ylindrica var. major community growing on the old Tongguanshanasteland (TIR and TIB) with a discarded period about 30 yearsad higher diversity indices than that growing on the Yangshan-

Fig. 2. Shannon indices (H′) of nifH gene from rhizosphere tailings (dashed bars) a

Numbered positions refer to the excised bands; texts in each lane indicate the typeof tailing samples shown in Table 1.

chong wasteland (YISR, YIMR, YISB and YIMB) with a discardedperiod of 19 years (Fig. 2), and the diversity indices of free-livingnitrogen-fixers displayed the following order: TIR > YIMR > YISRin rhizosphere tailing samples and TIB > YIMB > YISB in non-rhizosphere samples (Fig. 2).

The diversity indices of rhizosphere and non-rhizosphere tailingsamples increased with plant colonization period for I. cylindricavar. major and H. ramosissimum in Yangshanchong mine wasteland(Fig. 2), namely that the samples from plant community in maturephase presented higher diversity of free-living nitrogen-fixers thanthat from young phase plant community, whereas a contrary resultwas found for Z. sinica (Fig. 2).

In Yangshanchong wasteland, nitrogen-fixers in rhizosphereand non-rhizosphere tailings from I. cylindrica var. major commu-nity presented lower diversity indices than that in tailings fromH. ramosissimum, C. dactylon, Z. sinica and M. floridulus communi-ties; moreover, a lower diversity was also found in I. cylindrica var.major rhizosphere compared with C. dactylon and M. sinensis in old

Tongguanshan wasteland.

These results indicated that plant species, the age of plantcommunity and discarded period of wastelands could affect the

nd non-rhizosphere tailings (grid bars). For sample description, see Table 1.

J. Zhan, Q. Sun / Microbiological Research 167 (2012) 157– 165 161

Table 3Physico-chemical properties of mine wastelands.

Wasteland of coppermine tailings studied

Types of plantcommunities

pH EC (�S cm−1) AP(mg kg−1)

TN(mg kg−1)

LOI(mg kg−1)

AvailableCu(mg kg−1)

AvailableZn(mg kg−1)

Old Tongguanshan I. cylindrica var.major community

7.28 ± 0.37 1181.67 ± 692.69 1.21 ± 0.97 443.39 ± 134.19 4.23 ± 1.12 15.36 ± 4.25 9.33 ± 3.12

Yangshanchong I. cylindrica var.major community

7.73 ± 0.04 598.00 ± 121.67 2.20 ± 1.04 122.25 ± 36.60 1.28 ± 1.64 22.61 ± 7.45 13.80 ± 2.90

M. floriduluscommunity

7.69 ± 0.12 665.33 ± 170.04 1.26 ± 0.43 60.95 ± 8.34 0.81 ± 0.07 26.08 ± 4.64 8.19 ± 2.47

H. ramosissimumcommunity

8.11 ± 0.11 340.00 ± 47.68 0.76 ± 0.15 118.76 ± 15.43 1.09 ± 0.03 20.61 ± 6.44 9.62 ± 2.27

Z. sinicacommunity

7.96 ± 0.10 333.00 ± 73.75 1.39 ± 0.54 65.66 ± 25.89 0.33 ± 0.21 24.56 ± 6.96 9.98 ± 2.14

C. dactylon 7.79 ± 0.07 484.08 ± 103.29 0.19 ± 0.09 33.48 ± 11.57 2.12 ± 0.48 126.09 ± 15.18 24.40 ± 5.84

V AP: av

ds

3fi

tpoTfise2canoapf

Ffibb

community

alues are given as means ± standard error of the means; EC: electric conductivity;

iversity of free-living nitrogen-fixing microorganisms in rhizo-phere and non-rhizosphere tailings.

.3. Principal component analysis and cluster analysis of DGGEngerprints

To characterize the nitrogen-fixing microbial community struc-ure in rhizosphere and non-rhizosphere tailings with differentlant communities and different sampling wastelands, PCA basedn the DGGE banding patterns of nitrogen-fixers is shown in Fig. 3.he significant effect of plant species on the structures of nitrogen-xing microbial communities can be seen in PCA plot and theamples from two wastelands presented distinct distribution. How-ver, the percentage of the variance attributed to components 1 and

in PCA was only 18.0% and 13.4%, respectively, on account of theomplexity of influential factors. Moreover, the UPGMA clusteringnalysis was used to analyse the nitrogen-fixing microbial commu-ity similarity among different tailing samples (Fig. 4). Clustering

f the DGGE profiles revealed that there were very large differencesmong the nitrogen-fixing microbial communities in tailing sam-les from different plant communities. The greatest difference wasound between the samples from C. dactylon community (YCB and

ig. 3. Principal component analysis (PCA) of DGGE banding patterns of nitrogen-xing microbial communities in wasteland samples. Tailing samples are representedy different types of symbols, empty symbols: rhizosphere samples (-R), full sym-ols: non-rhizosphere samples (-B) (shown in Table 1).

ailable phosphorus; TN: total nitrogen; LOI: loss of ignition.

YCR) and all the other samples. The profiles of YCB and YCR belongto a single cluster. The profiles of the samples from Tongguanshanwasteland presented high similarities. In Yangshanchong waste-land, the rhizosphere (YMR) and non-rhizosphere (YMB) samplesfrom M. floridulus community were separated from other sam-ples from I. cylindrica var. major, H. ramosissimum and Z. sinicacommunities; for H. ramosissimum and Z. sinica community withdifferent development phases, the profiles of non-rhizosphere sam-ples (YZSB and YZMB, YHSB and YHMB) were divided into eachcluster, respectively, whereas the rhizosphere samples presenteddifferent nitrogen-fixing microbial community structure from non-rhizosphere samples for the same plant community; moreover,a low similarity of non-rhizosphere samples under I. cylindricavar. major was shown between young phase (YISB) and maturephase (YIMB). It also can be seen that the non-rhizosphere samples(YZMB, YZSB, YHMB, YHSB, YIMB) and rhizosphere (YZMR, YHMR,YHSR, YIMR) samples in Yangshanchong wasteland were inclinedto be in one cluster, respectively.

The clusters in the UPGMA dendrogram corresponded to thesegroups in PCA, which indicated the main effects of physico-chemical properties of wastelands and plant species on thestructure of free-living nitrogen-fixing microbial community inmine wastelands.

3.4. Phylogenetic analysis

A total of 52 different band positions could be identified in DGGEfingerprints, which were retrieved, followed by PCR, cloning and

sequencing. As a result, 37 sequences were got from DGGE gel whileremaining 15 bands were not sequenced successfully.

With BLAST-N analyses, 37 nifH gene fragments showed84–100% similarity with known sequences; and 36 bands (97.3%)

Percent Similarity

TCRTMRTIBTIRYMRYISRYISBYIMRYHSRYHMRYZMRYIMBYHSBYHMBYZSBYZMBYZSRYMBYCRYCB

4 20 36 52 68 84 100

Fig. 4. Cluster analysis based on the diversity of nifH genes in tailing samples. Thedetailed illumination of samples is shown in Table 1.

162 J. Zhan, Q. Sun / Microbiological Research 167 (2012) 157– 165

Band 32 Uncultured bacterium (FJ916072)

Band 36 Uncultured bacterium (DQ480885)

Uncultured bacterium (AJ716382) Mesorhizobium loti (AB367742)

Bradyrhizobium sp. (AB079616) Uncultured soil bacterium (DQ776510)

Band 22 Band 35

Uncultured bacterium (EU048053) Uncultured bacterium (EU048087)

Band 28 Bradyrhizobium sp. (AM110702)

Band 17 Bradyrhizobium elkanii (EF153399)

Band 1Band 15

Uncultured bacterium (AB471096) Uncultured bacterium (EU912866)

Uncultured bacterium (AB471072) Band 24

Band 25 Band 16 Band 21

Beijerinckia indica subsp. lacticogenes (AJ563939) Bradyrhizobium japonicum (GQ289562)

Sphingomonas azotifigens (AB217474) Band 31

Uncultured microorganism (DQ481379) Bradyrhizobium japonicum (GQ289565)

Uncultured bacterium (EF583588) Band 37

Band 34 Band 33

Burkholderia xenovorans (EF158805) Burkholderia xenovorans (CP000271)

Band 29 Herbaspirillum sp. (AB196476)

Uncultured bacterium (EF988491) Uncultured bacterium (EF988620)

Band 27 Band 10

Band 7 Uncultured soil bacterium (FJ008489) Band 30

Beta proteobacterium (EF626686) Band 26

Filamentous thermophilic cyanobacterium (DQ471425) Phormidium sp.(AF227927)

Leptolyngbya sp. (AY768415) Band 4 Band 5 Uncultured bacterium (EF988349)

Microcoleus sp. (AY768420) Uncultured bacterium (AB266355)

Band 11 Cyanothece sp. (CP001344)

Uncultured bacterium (DQ520379) Band 2

Uncultured bacterium (AJ716380) Band 9 Uncultured cyanobacterium (EF408200)

Band 3 Band 23

Band 14Band 12 Band 6

Band 18 Band 8

Band 19 Band 13 Band 20

100

100

99

9965

100

100

6167

65

55

100

71

100

99

9799

99

8651

68

59

57

99

8699

99

55

63

99

7291

90

76

59

51

89

64

63

58

65

0.02

α

β

Prot

eoba

cter

ia

Cyan

obac

teria

F nifH DB e nump ution.

wtxtbgT

ig. 5. Neighbour-joining tree depicting the polygenetic relationships among the

ank accession numbers. DGGE bands detected in this study are given in bold. Thseudoreplications. The values below 50 are not shown. The scale indicates substit

ere closely related to nifH genes of uncultured bacteria or uncul-ured cyanobacteria, and one band was affiliated with Burkholderiaenovorans (91% similarity). Consequently, the closest relatives and

he closest relatives of known genus corresponding to the DGGEands were both subjected to cluster analyses with sequencesenerated from DGGE gels by neighbour-joining method (Fig. 5).wenty-two of 37 sequences clustered in the Proteobacteria, and

GGE sequences in this study. Species or strain names are followed by their Gen-bers shown next to each bifurcation are bootstrap percent values based on 5000

15 sequences grouped within the Cyanobacteria. Within the Pro-teobacteria, 16 and 6 sequences belonged to the �-Proteobacteriaand �-Proteobacteria, respectively (Fig. 5).

All samples have a �-Proteobacteria nifH gene fragment (Band10), and most samples have a cyanobacteria nifH gene fragment(Band 18) (Figs. 1 and 5). The non-rhizosphere samples showeda higher number of cyanobacteria nifH genes than rhizosphere

J. Zhan, Q. Sun / Microbiological Re

Percent Similarity

TI

YI

YM

YC

YH

YZ

70605040 100 9080

Fig. 6. Cluster analysis of tailing samples from different pioneer plant communitiesbased on the physico-chemical properties of samples. TI represents samples from I.caa

srsnd

4

4m

dftssvsi

ascYtwcsimpipc(ccpcsgPteb

ylindrica var. major community in Tongguanshan wasteland; YI, YM, YC, YH and YZre samples from I. cylindrica var. major, M. floridulus, C. dactylon, H. ramosissimumnd Z. sinica community, respectively.

amples. In Yangshanchong wasteland of copper mine tailings, thehizosphere and non-rhizosphere tailing samples from H. ramosis-imum community growing on wet areas have more cyanobacteriaifH genes than samples from I. cylindrica var. major community, C.actylon and M. floridulus community growing on dry areas.

. Discussion

.1. Factors causing changes of free-living nitrogen-fixingicrobial community diversity

Although nitrogen-fixing microorganisms are highly adapted toifferent environmental conditions and considered to be importantor the nitrogen input to soil, they are rarely dominant in terres-rial ecosystems (Wartiainen et al. 2008; Coelho et al. 2009) andusceptible to environmental condition. In this study, the diver-ity of nitrogen-fixing microbial community in wasteland samplesaried with the physico-chemical properties of wastelands, plantpecies, and the development period of plant community, and wasnfluenced by plant rhizosphere.

In the old Tongguanshan wasteland with high nutrient contentsnd less toxicity of heavy metals, the nitrogen-fixers in rhizo-phere and non-rhizosphere samples under I. cylindrica var. majorommunity (TIB and TIR) displayed higher diversity than that inangshanchong wasteland. It also can be seen from PCA and clus-er analysis (Figs. 3 and 4) that the samples in Tongguanshanasteland presented distinct structure of nitrogen-fixing microbial

ommunity from that in Yangshanchong wasteland and the diver-ity of nitrogen-fixers in wastelands samples increased with themprovement of wasteland condition along with the restoration of

ine wastelands (Fig. 2). The cluster analysis of physico-chemicalroperties of mine wastelands from different plant communities

s shown in Fig. 6, indicating the distinct difference between sam-les from two tailing wastelands, which was consistent with theluster analysis of microbial samples based on the DGGE profileFig. 4), further confirming the determinant effect of the nutrientontent and pollution on the structure of nitrogen-fixing microbialommunity. Tan et al. (2003) suggested that the environmentalarameters, especially N content had a strong influence on theommunity of root-colonizing diazotrophs, rather than the plantpecies. Sato et al. (2009) reported that the diversity and phylo-enetic composition of nifH genes depended on soil properties.

revious work also confirmed the key effect of C and N content onhe nitrogen-fixing bacterial community (Poly et al. 2001b; Zhangt al. 2006; Coelho et al. 2008, 2009). Moreover, nitrogen fixationy free-living heterotrophic bacteria was found to be inhibited in

search 167 (2012) 157– 165 163

metal contaminated soil (McGrath et al. 1995; Mårtensson andTorsrensson 1996). The population of free-living nitrogen-fixerswas significantly affected in contaminated soil with the heavymetal content exceeded the limit established by the CEC Directive(1986), undergoing a decrease of about 80% (Oliveira and Pampulha2006; Oliveira et al. 2009).

In this study, significant difference in the free-living nitrogen-fixing microbial community can be seen due to the type ofplant community (Fig. 3 and Fig. 4). Moreover, the diversity ofnitrogen-fixers in rhizosphere and non-rhizosphere samples underHemicryptophytes with stolon (C. dactylon) was generally higherthan that of tailings under Geophytes with rhizome (I. cylindricavar. major and H. ramosissimum) growing on the Yangshanchongmine wasteland. In addition, the diversity of rhizosphere nitrogen-fixers from C. dactylon was also higher than that of samples fromI. cylindrica var. major in the old Tongguanshan mine wasteland.These results suggested the significant effect of plant species onthe diversity and structure of nitrogen-fixers communities in thetailings. It has been already shown that composition, number anddistribution of nitrogen-fixers were associated with plant species(Tan et al. 2003; Diallo et al. 2004; Duc et al. 2009). According tothese results, the physico-chemical properties of wastelands andplant species were proved to be the dominant factors determiningthe structure of free-living nitrogen-fixing microbial community intailing wastelands.

In addition, in Yangshanchong wasteland, for the samplesfrom pioneer plant communities (I. cylindrica var. major andH. ramosissimum) in young and mature phases, the latter dis-played high diversity of free-living nitrogen-fixers. The vegetationcover increased with plant colonization time which may decreasefluctuations in temperature and moisture of the upper layer ofwastelands, and improve the C and N contents of wastelands.Tscherko et al. (2005) thought that the increase in vegetation coverdecreased heat stress and dryness at the soil surface, favoringmicrobial growth and activity. In the case of Z. sinica, the diversityof nitrogen-fixers in rhizosphere and non-rhizosphere tailing sam-ples decreased with plant colonization time, probably because oflow moisture and low contents of C and N. Indeed, the water contentof samples under Z. sinica was significantly lower than that of sam-ples under I. cylindrica var. major and H. ramosissimum, especiallyin the area early colonized by Z. sinica.

It can also be seen from cluster analysis that the non-rhizospheresamples from pioneer plant communities in Yangshanchong waste-land had high similarities, the same as rhizosphere samples (Fig. 4).For H. ramosissimum and Z. sinica community, the rhizosphereeffect of plant led to obvious change of free-living nitrogen-fixingmicrobial community (Fig. 4). Regardless of plant species, thenitrogen-fixers communities in the non-rhizosphere tailings hadhigher diversity than those in rhizosphere tailings in Yangshan-chong mine wasteland (Figs. 1 and 2). Similar findings are reportedby Duc et al. (2009) who showed that nifH gene sequences ofthe bulk soil were higher than those of the rhizosphere. Thehigher diversity of nifH gene sequences in non-rhizosphere sam-ples may result from a higher diversity of the microhabitat, morefluctuating growth conditions in non-rhizosphere samples (Ducet al. 2009) and the presence of a higher number of nitrogen-fixing cyanobacteria. In this study, band analysis of DGGE profilesindicated the non-rhizosphere tailing samples presented higherdiversity of cyanobacteria nifH gene fragments than rhizospheretailing samples. Nevertheless, contrary results have been observedby Coelho et al. (2009) who suggested that the nitrogen-fixingpopulations in the rhizospheres of sorghum were more diverse

than those in bulk soil for the stimulation of rhizosphere exu-dates to diazotrophic growth, and Wartiainen et al. (2008) andSato et al. (2009) found that there was no remarkable differencein nifH gene diversity between the rhizosphere and bulk soil. The

1 ical Re

rosi(bag

4i

nar�Ar2

,misanCbh

ccs

ntmefb(fbnlgweacD

sac(swpmwratt

64 J. Zhan, Q. Sun / Microbiolog

hizosphere samples from Z. sinica community with different devel-pment stages (YZSR and YZMR) presented different diversity andtructure of nitrogen-fixing microbial community (Figs. 2 and 4)n the case of relative high similarity of non-rhizosphere samplesYZSB and YZMB) (Fig. 4). The diazotrophic growth can be affectedy the rhizosphere exudates (Soares et al. 2006) and the amountnd composition of rhizosphere exudates varies with plant species,rowth stage and other environmental factors (Hütsch et al. 2002).

.2. Structure of free-living nitrogen-fixing microbial communityn wasteland samples

The amplification of nifH gene from the rhizosphere andon-rhizosphere DNA extracts was performed by nested PCRpproach proved to be necessary. Free-living nitrogen-fixers inhizosphere and bulk soil mainly included Proteobacteria (�-, �-,-, �-Proteobacteria, etc.), Firmicutes, Spirochetes, Cyanobacteria andrchaea, and Proteobacteria were commonly dominant in soil envi-onment (Deslippe et al. 2005; Zhang et al. 2006; Wartiainen et al.008; Coelho et al. 2008).

In our study, there were only nifH gene sequences grouped in �- �-Proteobacteria and Cyanobacteria (Fig. 5). For �-Proteobacteria,

ost nifH sequences were affiliated with Bradyrhizobium belong-ng to slow-growing rhizobia, which were moderately adapted toemi arid conditions (Kahindi et al. 1997). Bradyrhizobium has beenlready observed as an active nitrogen-fixing bacterium to colo-ize roots of a broad range of nonlegume (Chaintreuil et al. 2000;oelho et al. 2008), and both free-living and root-associated sym-iotic Bradyrhizobium were able to fix nitrogen in autotrophic andeterotrophic conditions (Kahindi et al. 1997).

No nifH gene sequence of �-, �-Proteobacteria, Firmicutes, Spiro-hetes and Archaea was detected, and this may be due to the biasaused by preferential amplification, or that they were present inuch a low number to be undetectable (Wartiainen et al. 2008).

The wastelands of copper mine tailings studied showed lowutrient contents and poor soil structure, and were subjected toemperature and moisture fluctuation and the toxicity of heavy

etals (Table 3), whose condition is similar to deserts (Chowdhuryt al. 2009). In the Jaisalmer desert of India, the nifH gene sequencesrom Proteobacteria in rhizosphere and bulk soils were prevailing,ut that from Firmicutes, Cyanobacteria and Archaea was absentChowdhury et al. 2009). In our study, the nifH gene sequencesrom Cyanobacteria were abundant (15 bands, Fig. 5), distributingoth in rhizosphere and non-rhizosphere tailings. The adaptation ofitrogen-fixing cyanobacteria to the condition of poor mine waste-

and were likely related to the existence of cryptogamic crustsrowing on the surface of wastelands of copper mine tailings, whichere composed of cyanobacteria, lichen, bacteria, fungi and moss,

tc. (Néel et al. 2003; Sun et al. 2004). Studies in desert, plateau andrctic area have demonstrated that there are many nitrogen-fixingyanobacteria in biological soil crusts (Yeager et al. 2004, 2007;eslippe et al. 2005).

Different banding patterns of nifH gene sequences were pre-ented in the DGGE profile (Fig. 1), however, the rhizospherend non-rhizosphere tailings from different predominant plantommunities showed similar nitrogen-fixing species compositionFig. 5), suggesting that the dominant groups were relativelytable in the mine wastelands investigated. The copper miningasteland had been subjected to poor nutrient and heavy metalollution over a long time period, and the nitrogen-fixers existeday be species that were adapted to special habitats of mineastelands. In this study, most nifH sequences were not closely

elated to any nitrogen-fixing microorganisms of known genus,nd 54% sequences exhibited less than 90% nucleotide acid iden-ity with known sequences in the database, which indicated thatheir identities were uncertain, and the closest relatives of 36

search 167 (2012) 157– 165

bands were uncultured microorganisms without fully understand-ing on their function. These results suggested that most of thesenifH-containing microorganisms detected in this study might beunculturable, and that the nifH gene sequences in mine waste-lands investigated are unique and may represent novel sequencesof nitrogen-fixing microorganisms.

In conclusion, we suggested that the diversity of free-living nitrogen-fixing microorganisms in rhizosphere and non-rhizosphere tailings was mainly influenced by wastelandsproperties and plant species, especially the accumulation of nutri-ent and the reduction of heavy metal toxicity along with thecolonization of plant community. The old Tongguanshan waste-land with a longer discarded period and soil formation presentedhigher diversity of free-living nitrogen-fixers in the rhizosphereand non-rhizosphere tailings from I. cylindrica var. major commu-nity than Yangshanchong wasteland, and this demonstrated thatthe improvement of wasteland properties as a result of the increas-ing age of soil development may enhance the diversity of free-livingnitrogen-fixers. Furthermore, the non-rhizosphere tailings gen-erally had higher diversity of nitrogen-fixers than rhizospheretailings likely due to the diversified microhabitat of the former. Sim-ilar free-living nitrogen-fixing species composition were detectedin rhizosphere and non-rhizosphere tailings, Proteobacteria (�-, �-Proteobacteria) and Cyanobacteria included, and most sequenceswere closely related to uncultured microorganisms which mayrepresent novel sequences of nitrogen-fixing microorganisms. Thecharacterizing of these nitrogen-fixers, as well as the tolerancemechanism involved would be investigated in further study.

Acknowledgement

This work was financially supported by The Ministry of Envi-ronmental Protection of the People’s Republic of China (No.201009041-02).

References

Aquilanti L, Mannazzu I, Papa R, Cavalca L, Clementi F. Amplified ribosomal DNArestriction analysis for the characterization of Azotobacteraceae: a contributionto the study of these free-living nitrogen-fixing bacteria. J Microbiol Methods2004;57:197–206.

Beneduzi A, Peres D, da Costa PB, Zanettini MHB, Passaglia LMP. Genetic and phe-notypic diversity of plant-growth-promoting bacilli isolated from wheat fieldsin southern Brazil. Res Microbiol 2008;159:244–50.

Chaintreuil C, Giraud E, Prin Y, Lorquin J, Ba A, Gillis M, et al. Photosynthetic Bradyrhi-zobia are natural entophytes of the African wild rice Oryza breviligulata. ApplEnviron Microbiol 2000;66:5437–47.

Chowdhury SP, Schmid M, Hartmann A, Tripathi AK. Diversity of 16S-rRNA and nifHgenes derived from rhizosphere soil and roots of an endemic drought tolerantgrass, Lasiurus sindicus. Eur J Soil Biol 2009;45:114–22.

Coelho MRR, de Vos M, Carneiro NP, Marriel IE, Paiva E, Seldin L. Diversity ofnifH gene pools in the rhizosphere of two cultivars of sorghum (Sorghumbicolor) treated with contrasting levels of nitrogen fertilizer. FEMS MicrobiolLett 2008;279:15–22.

Coelho MRR, Marriel IE, Jenkins SN, Lanyon CV, O’Donnell AG. Molecular detec-tion and quantification of nifH gene sequences in the rhizosphere of sorghum(Sorghum bicolor) sown with two levels of nitrogen fertilizer. Appl Soil Ecol2009;42:48–53.

Commission of the European Communities (CEC). Council directive on the protectionof the environment, and in particular of the soil, when sewage sludge is used inagriculture. Offic J Eur Commun 1986, L181/6.

Deslippe JR, Egger KN, Henry GHR. Impacts of warming and fertilization on nitrogen-fixing microbial communities in the Canadian High Arctic. FEMS Microbiol Ecol2005;53:41–50.

Diallo MD, Willems A, Vloemans N, Cousin S, Vandekerckhove TT, de Lajudie P, et al.Polymerase chain reaction denaturing gradient gel electrophoresis of the N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanitesaegyptiaca in the dryland part of Senegal. Environ Microbiol 2004;6:400–15.

Duc L, Noll M, Meier E, Burgmann H, Zeyer J. High diversity of diazotrophs in the

forefield of a receding alpine glacier. Microb Ecol 2009;57:179–90.

Hsu SF, Buckley DH. Evidence for the functional significance of diazotroph commu-nity structure in soil. Int Soc Microb Ecol 2009;3:124–36.

Hütsch BW, Augustin J, Merbach W. Plant rhizodeposition – an important source forcarbon turnover in soils. J Plant Nutr Soil Sci 2002;165:397–407.

ical Re

K

M

M

N

N

O

O

P

P

S

S

S

J. Zhan, Q. Sun / Microbiolog

ahindi JHP, Woomer P, George T, de Souza Moreira FM, Karanja NK, Giller KE. Agri-cultural intensification, soil biodiversity and ecosystem function in the tropics:the role of nitrogen-fixing bacteria. Appl Soil Ecol 1997;6:55–76.

årtensson AM, Torsrensson L. Monitoring sewage sludge using heterotrophicnitrogen fixing microorganisms. Soil Biol Biochem 1996;28:1621–30.

cGrath SP, Chaudr AM, Giller KE. Long-term effects of metals in sewage sludge onsoils, microorganisms and plants. J Ind Microbiol 1995;14:94–104.

anjing Institute of Soil Science, Chinese Academy of Sciences. Soil physical andchemical analysis. Shanghai: Shanghai Science and Technology Press; 1978.

éel C, Bril H, Courtin-Nomade A, Dutreuil JP. Factors affecting natural develop-ment of soil on 35-year-old sulphide-rich mine tailings. Geoderma 2003;111:1–20.

liveira A, Pampulha ME. Effects of long-term heavy metal contamination on soilmicrobial characteristics. J Biosci Bioeng 2006;102:157–61.

liveira A, Pampulha ME, Neto MM, Almeida AC. Enumeration and characterizationof arsenic-tolerant diazotrophic bacteria in a long-term heavy-metal-contaminated soil. Water Air Soil Pollut 2009;200:237–43.

oly F, Monrozier LJ, Bally R. Improvement in the RFLP procedure for studying thediversity of nifH genes in communities of nitrogen fixers in soil. Res Microbiol2001a;152:95–103.

oly F, Ranjard L, Nazaret S, Gourbière F, Monrozier LJ. Comparison of nifH gene poolsin soils and soil microenvironments with contrasting properties. Appl EnvironMicrobiol 2001b;67:2255–62.

ato A, Watanabe T, Unno Y, Purnomo E, Osaki M, Shinano T. Analysis of diver-sity of diazotrophic bacteria associated with the rhizosphere of a tropical arbor,Melastoma malabathricum L. Microb Environ 2009;24:81–7.

imonet P, Grojean MC, Misra AK, Nazaret S, Cournoyer B, Normand P. Frankia genus-specific characterization by polymerase chain reaction. Appl Environ Microbiol

1991;57:3278–86.

oares RA, Roesch LFW, Zanatta G, de Oliveira Camargo FA, Passaglia LMP. Occur-rence and distribution of nitrogen fixing bacterial community associated withoat (Avena sativa) assessed by molecular and microbiological techniques. ApplSoil Ecol 2006;33:221–34.

search 167 (2012) 157– 165 165

Sun QY, An SQ, Yang LZ, Wang ZS. Chemical properties of the upper tailings beneathbiotic crusts. Ecol Eng 2004;23:47–53.

Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Anal-ysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9.

Tan Z, Hurek T, Reinhold-Hurek B. Effect of N-fertilization, plant genotype and envi-ronmental conditions on nifH gene pools in roots of rice. Environ Microbiol2003;5:1009–15.

Tian SN, Sun QY, Wang ZF, Peng SL, Xia HP. Plant colonization on copper tailingsand the change of the physico-chemical properties of substrate in Tongling City,Anhui Province. Resour Environ Tangtze Basin 2005;14:88–93.

Tscherko D, Hammesfahr U, Zeltner G, Kandeler E, Böcker R. Plant succession and rhi-zosphere microbial communities in a recently deglaciated alpine terrain. BasicAppl Ecol 2005;6:367–83.

Unkovich M, Baldock J. Measurement of asymbiotic N2 fixation in Australian agri-culture. Soil Biol Biochem 2008;40:2915–21.

Wartiainen I, Eriksson T, Zhang W, Rasmussen U. Variation in the active diazotrophiccommunity in rice paddy – nifH PCR-DGGE analysis of rhizosphere and bulk soil.Appl Soil Ecol 2008;39:65–75.

Widmer F, Shaffer BT, Porteous LA, Seidleret RJ. Analysis of nifH gene pool complexityin soil and litter at a Douglas fir forest site in Oregon cascade mountain range.Appl Environ Microbiol 1999;65:374–80.

Yeager CM, Kornosky JL, Housman DC, Grote EE, Belnap J, Kuske CR. Diazotrophiccommunity structure and function in two successional stages of biological soilcrusts from the Colorado Plateau and Chihuahuan Desert. Appl Environ Micro-biol 2004;70:973–83.

Yeager CM, Kornosky JL, Morgan RE, Cain EC, Garcia Pichel F, Housman DC, et al. Threedistinct clades of cultured heterocystous cyanobacteria constitute the dominantN2-fixing members of biological soil crusts of the Colorado Plateau, USA. FEMS

Microbiol Ecol 2007;60:85–97.

Zhang Y, Li D, Wang H, Xiao HM, Liu XD. Molecular diversity of nitrogen-fixingbacteria from the Tibetan Plateau, China. FEMS Microbiol Lett 2006;260:134–42.

Zhou JZ, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. ApplEnviron Microbiol 1996;62:316–22.