R E S EA RCH L E T T E R

Differential distribution and abundance of diazotrophicbacterial communities across different soil niches using a

gene-targeted clone library approach

Basit Yousuf1,2, Raghawendra Kumar1,2, Avinash Mishra1,2 & Bhavanath Jha1,2

1Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute (CSIR-CSMCRI), Bhavnagar, Gujarat,

India; and 2Academy of Scientific and Innovative Research (AcSIR), CSIR, New Delhi, India

Correspondence: Avinash Mishra,

Discipline of Marine Biotechnology and

Ecology, CSIR-Central Salt and Marine

Chemicals Research Institute (CSIR-CSMCRI),

G. B. Marg, Bhavnagar, Gujarat, 364002

India.

Tel.: +91 278 2567760 Ext. 6260;

fax: +91 278 2570885;

e-mail: avinash@csmcri.org

Received 11 July 2014; revised 27 August

2014; accepted 31 August 2014.

DOI: 10.1111/1574-6968.12593

Editor: Tim Daniell

Keywords

diazotrophs; clone library; microbial diversity;

nifH; saline soil.

Abstract

Diazotrophs are key players of the globally important biogeochemical nitrogen

cycle, having a significant role in maintaining ecosystem sustainability. Saline

soils are pristine and unexplored habitats representing intriguing ecosystems

expected to harbour potential diazotrophs capable of adapting in extreme con-

ditions, and these implicated organisms are largely obscure. Differential occur-

rence of diazotrophs was studied by the nifH gene-targeted clone library

approach. Four nifH gene clone libraries were constructed from different soil

niches, that is saline soils (low and high salinity; EC 3.8 and 7.1 ds m�1), and

agricultural and rhizosphere soil. Additionally, the abundance of diazotrophic

community members was assessed using quantitative PCR. Results showed

environment-dependent metabolic versatility and the presence of nitrogen-fix-

ing bacteria affiliated with a range of taxa, encompassing members of the

Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacte-

ria, Cyanobacteria and Firmicutes. The analyses unveiled the dominance of

Alphaproteobacteria and Gammaproteobacteria (Pseudomonas, Halorhodospira,

Ectothiorhodospira, Bradyrhizobium, Agrobacterium, Amorphomonas) as nitrogen

fixers in coastal–saline soil ecosystems, and Alphaproteobacteria and Betaproteo-

bacteria (Bradyrhizobium, Azohydromonas, Azospirillum, Ideonella) in agricul-

tural/rhizosphere ecosystems. The results revealed a repertoire of novel

nitrogen-fixing bacterial guilds particularly in saline soil ecosystems.

Introduction

The recycling of nitrogen contributes substantially to

nutrient fluxing and sustainable soil fertility in the terres-

trial ecosystem (Hsu & Buckley, 2009; Cavalcante et al.,

2012). Around half of the annual nitrogen is fluxed into

the biosphere (Vitousek et al., 1997), and natural (Cleve-

land et al., 1999) and agricultural ecosystems (Peoples &

Craswell, 1992) by biological nitrogen fixation, which

involves conversion of N2 to ammonia (NH3). This com-

plex process is catalysed by the nitrogenase reductase

enzyme, mediated through certain groups of bacteria/ar-

chaea (diazotrophs) in symbiotic and associative or under

free-living conditions (Zehr et al., 2003). This enzyme

consists of two component metalloproteins, the iron (Fe)

protein (encoded by nifH) and the molybdenum–iron

(Mo–Fe) protein (encoded by nifD and nifK) (Zehr et al.,

2003). Additionally, this enzyme requires several other

additional genes, such as nifE, nifN, nifX, nifQ, nifW,

nifV, nifA, nifB, nifZ and nifS, which act coordinately

for functioning of the active enzyme (Lee et al., 2000;

Masepohl et al., 2002).

The nifH gene is widely distributed among phylogeneti-

cally diverse bacteria and archaea (Poly et al., 2001;

Dixon & Kahn, 2004). The gene is commonly used for

the study of phylogeny, diversity and abundance of both

cultured and uncultivated organisms from multiple envi-

ronments (Zehr et al., 2003), as its protein sequence is

well conserved compared with other genes of the nif

operon. The relationship among bacteria based on the

sequence divergences of this gene has been reported to be

congruent with 16S rRNA gene phylogeny with some

FEMS Microbiol Lett && (2014) 1–9 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

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exceptions (Ueda et al., 1995; Borneman et al., 1996;

Zehr et al., 2003). The nifH gene has sufficient variation

to detect shifts in the community structure of nitrogen-

fixers in ecosystems under varying physicochemical

characteristics and soil types (Bagwell et al., 2002; Pere-

ira-e-Silva et al., 2011), as each habitat selects compatible

different groups of nitrogen-fixing organisms (Zehr et al.,

2003). Changes in different environmental factors, such

as soil moisture, oxygen, pH, electrolytic conductivity,

and carbon, nitrogen and sulphur contents have been

reported to influence nitrogen fixation in soils (Hsu &

Buckley, 2009).

A few culture-independent studies have been per-

formed on the functional diversity of nitrogen-fixing

microbial communities in moderate, extreme, terrestrial,

bulk and rhizosphere soil environments, such as rice, for-

est, grasses, soybean and sediments (Hirano et al., 2001;

Chowdhury et al., 2009; Xiao et al., 2010; Orr et al.,

2011, 2012; Cavalcante et al., 2012), but has not been

equally addressed from saline soil ecosystems (Keshri

et al., 2013). Keshri et al. (2013) focused on only one sal-

ine soil and also reported fewer clones (51) and opera-

tional taxonomic units (OTUs; 20). Saline soil ecosystems

represent intriguing ecological niches, where anaerobic or

microaerophilic conditions are expected to prevail. These

niches are widely distributed in arid and semiarid regions,

occupying 6% of the total global land surface and 2% of

the geographical area in India (Yadav, 2003).

In this study, the comparative molecular analysis of

diazotrophs was performed by targeting key nitrogenase

reductase enzymes of the biogeochemical nitrogen cycling

pathway from coastal–saline, agricultural and rhizosphere

soils. These soil niches were previously assessed for

chemolithoautotrophic metabolism, using gene-targeted

metagenomics (Yousuf et al., 2012a, b, 2014). The aim of

the present work was to broaden our view of the diversity

and abundance of nitrogen-fixing bacterial communities

and their comparative distribution among these environ-

mental niches.

Materials and methods

Soil samples and physicochemical

characteristics

Four distinct sites, comprising three bulk soil types,

namely low saline (SS1), high saline (SS2), agriculture

(AS) and one rhizospheric (RS) soil, were selected along

the Arabian Sea coast, Gujarat, India, and the composite

soil samples were collected in triplicate (Supporting

Information, Data S1). Physicochemical characteristics

(Table S1) were analysed as described previously (Yousuf

et al., 2012a, b, 2014).

DNA extraction, gene amplification and

construction of clone libraries

Soil DNA was extracted in triplicate from each soil

sample (Yousuf et al., 2012b) and the nifH gene was

amplified, using a degenerate primer pair PolF and PolR

(50-TGCGAYCCSAARGCBGACTC-30 and 50-ATSGCCATCATYTCRCCGGA-30; Poly et al., 2001). The nifH gene

amplicons were purified, cloned in pGEM-T Easy vector,

screened for correct insert size (360 bp) and positive

clones were sequenced (M/s Macrogen Inc., South Korea).

GenBank accession numbers

All the validated nifH gene sequences were deposited in

the GenBank database with accession numbers

KF861040–KF861509.

Alignment and phylogenetic reconstruction

The validated sequences were subjected to BLASTn

searches and the most similar sequences from GenBank

were retrieved for phylogenetic analysis (Altschul et al.,

1990). Multiple sequence alignment was performed by

Clustal Omega (Sievers et al., 2011) for the generation of

OTUs (phylotypes) using the program MOTHUR (Schloss

et al., 2009). Model selection analysis was conducted to

calculate the best-fit model of nucleotide substitution by

MEGA v.5.2 based on the lowest Bayesian Information

Criterion (Tamura et al., 2011). The evolutionary history

of all genes was inferred by maximum likelihood, using

the bootstrap resampling method with 500 bootstrap

replications.

Phylogenetic comparison and statistical

analysis

A sequence similarity cut-off of 95% (Jiang et al., 2009)

was used to define an OTU (phylotype) using MOTHUR

(Schloss et al., 2009). Jukes–Cantor evolutionary distance

matrices were calculated by DNADIST within PHYLIP version

3.2 (Felsenstein, 1989). The a-diversity indices (ACE and

Chao), OTUs, rarefaction curves, Shannon & Simpson

diversity indices and coverage were evaluated using MO-

THUR (Schloss et al., 2009). The datasets were also com-

pared for b-diversity, based on principal component

analysis (PCA), UNIFRAC significance and the P-test within

UNIFRAC (Lozupone & Knight, 2005; Lozupone et al.,

2006) to determine significantly different environments.

The interrelationship between environmental parameters,

diversity indices and distribution of taxonomic groups

was analysed by canonical correspondence analysis (CCA)

using PAST version 2.14 (Hammer et al., 2001).

FEMS Microbiol Lett && (2014) 1–9ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

2 B. Yousuf et al.

Quantitative PCR (qPCR)

Absolute quantification of diazotrophic bacteria in each

sample was carried out using a QuantiFast Kit (Qiagen)

with primer pair PolF and PolR, using the following PCR

programme: 95 °C for 5 min, and 35 cycles of 95 °C for

30 s, 55 °C for 30 s and 72 °C for 30 s. The experiments

were repeated three times, independently and the ampli-

fied product was run on 1.5% agarose gel to confirm the

expected size. The efficiency of qPCR was calculated, data

were analysed by the comparative CT method and copy

number of the targeted gene was determined (Yousuf

et al., 2012a).

Results

Functional community structure

Three clone libraries from each site were constructed to

determine the variation within sites (Fig. S1). Clone

libraries showed 92–96% similarity with each other, thus

indicating a very low variation within the site. The results

were supported by weighted UNIFRAC environmental clus-

tering analysis, which showed that bacterial communities

were not significantly different within the site (UNIFRAC

P = 0.9 for SS1, 0.9 for SS2, 0.7 for AS and 0.8 for RS,

whereas P-test P values were 0.9, 1.0, 0.5 and 0.9, respec-

tively).

The nifH clone libraries were constructed and totals of

122, 129, 100 and 119 clone sequences were obtained

from SS1, SS2, AS and RS, which yielded 50, 55, 42 and

32 unique phylotypes, respectively (Table 1). The richness

of the nifH gene was low in RS (0.26 OTUs per clone),

but high in SS1, SS2 and AS (0.40–0.42 OTUs per clone).

The library SS1 revealed the dominance of sequences

affiliated with nifH genes from Gammaproteobacteria (42

clones) followed by Alphaproteobacteria (16) and Delta-

proteobacteria (4) phylogenetic groups (Fig. 1). The dif-

ferent dominant genera encompassed by Proteobacteria

include Pseudomonas (15 phylotypes), Halorhodospira

halophila (13), filamentous Cyanobacteria (11), Bradyrhiz-

obium japonicum (8) and Ectothiorhodospira (7). Other

genera represented by few phylotypes included Heliobac-

terium modesticaldum (5), different species of Azospiril-

lum (5), Amorphomonas oryzae (4), Methylogaea (2),

Agrobacterium (3), Ideonella (1), Desulfovibrio (2) and

Desulfomicrobium (1). The SS2 clone library had a high

abundance of Alphaproteobacteria (59 clones) followed by

Gammaproteobacteria (26), whereas the most abundant

phylotypes that showed affiliation to cultured representa-

tives were Bradyrhizobium (15 clones), Rhizobium radiob-

acter (11), Amorphomonas oryzae (13) and Halorhodospira

halophila (12) (Fig. 1). The library also contained

phylotypes related to other genera such as Geoalkalibact-

er (2). These libraries sheltered a large pool of novel

nifH gene sequences, which showed low similarity to

the nifH gene harbouring cultured bacteria and clone

sequences from natural environments (Table S2). The

agricultural and rhizosphere soils were predominantly

represented by nifH gene sequences affiliated to Alpha-

proteobacteria (AS, RS; 47, 58 clones) and Betaproteobac-

teria (16, 37) (Fig. 1). The highly abundant nifH OTUs

showed affiliation with B. japonicum (AS, RS; 9, 22

clones), B. denitrificans (0, 5), Azohydromonas australica

(1, 25), Azospirillum zeae (4, 20), A. brasiliense (15, 4)

and Ideonella dechloratans (13, 11). A few clones were

related to Desulfovibrio gigas (RS, 2 clones), Methylocal-

dum szegediense (RS, 3), R. radiobacter (4, 2), Pseudo-

monas (5, 1), Dechloromonas sp. (RS, 1), Sinorhizobium

(4, 2), Halorhodospira halophila (1, 1) and Paenibacillus

(AS-7). About 69 and 56% of AS and RS OTUs,

respectively, showed higher nucleotide identity (94–99%) with the published GenBank sequences, although

only 10 and 20% of OTUs of SS1 and SS2 clone

libraries, respectively, showed high nucleotide identity

(94–97%). In AS and RS clone libraries, the Gammapro-

teobacteria, Deltaproteobacteria and Firmicutes phyloge-

netic groups were represented by few clones. It was

observed that Gammaproteobacteria dominated in saline

soil ecosystems (P < 0.01) while Betaproteobacteria

occurred prevalently (P < 0.01) in agricultural and rhi-

zosphere soils (Fig. 1). The majority of the nifH gene

sequences showed close affiliation to sequences from

uncultured organisms and revealed lower homology to

cultured representatives.

Abundance of functional nifH gene

The qPCR results (efficiency 97.32%; R2 = 0.9908)

showed a heterogeneous distribution of the nifH gene

among four sample sites (Fig. S2). The gene copies in rhi-

zospheric soil had significantly (P < 0.01) lower abun-

dance than in saline soil ecosystems. A trend towards a

significant (P < 0.01) increase in the abundance of nifH

gene copies per gram of soil was observed from rhizo-

spheric soil to saline soil (Table 1), except between SS2

and AS soil types.

Differentiation of diazotrophs based on a-

diversity indices

The nifH gene sequences showed high diversity at SS1,

SS2 and AS, and least diversity at RS (Fig. 1), as revealed

by parametric and nonparametric diversity indices

(Table 1). The diversity pattern was also supported by

rarefaction curves (Fig. S3), which inclined towards

FEMS Microbiol Lett && (2014) 1–9 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Diazotrophs in coastal saline soil ecosystems 3

saturation for SS1, SS2 and AS clone libraries, whereas

the curve almost levelled off for the RS library (Fig. S3).

The Venn diagram of nifH gene libraries provides com-

prehensive information on the differential distribution of

phylotypes, which showed the overlapping of nine, two

and 12 phylotypes among SS1 and SS2, SS1 and AS, and

AS and RS clone libraries, respectively (Fig. 2).

UNIFRAC and CCA

UNIFRAC-PCA analysis indicated the differential assemblages

of nifH gene sequences at all four habitats (UNIFRAC

P ≤ 0.03). The ordination diagram (Fig. S4) revealed vari-

ation between the data sets of agricultural (AS and RS)

and saline soils (SS1 and SS2), as they were separated on

the first axis that explains high variation (44.35%),

whereas the second axis did not show strong variation

(31.89%). UNIFRAC analyses revealed differential commu-

nity structure and diversity among different soil ecosys-

tems and were supported by P significance tests

(P < 0.001), when P-values have not been corrected for

multiple comparisons. The differentiation was supported

by a P value of 0.06, showing marginal differentiation,

when P-values have been corrected for multiple compari-

sons using the Bonferroni correction.

A triplot was generated to define the environmental

parameters for the dominance of microbial guilds across

Table 1. Biodiversity and predicted richness of nifH gene clone libraries

Soil

types

No. of

clones

Observed

OTUs*

Shannon–

Weiner (H)

Simpson

(1�D) Chao ACE Jackknife

Coverage

(%)

No. of

singletons

Copy

numbers per g soil

SS1 122 50 3.5 0.96 92.8 118.4 98.5 77 27 2.06 � 0.07 9 108

SS2 129 55 3.5 0.95 129.4 167.4 940 72 35 2.03 � 0.06 9 107

AS 100 42 3.4 0.96 81.4 77.9 85.04 76 24 1.33 � 0.09 9 107

RS 119 32 2.9 0.92 41.7 55 45 89 13 7.00 � 0.08 9 106

*OTUs for nifH clone libraries were determined at a 0.05 distance cut-off. Coverage, Shannon–Weiner (H), Simpson (1�D), Jackknife indices and

Chao & ACE richness estimators were calculated using the OTU data.

60 Alphaproteobacteriaa

50

BetaproteobacteriaGammaproteobacteriaDeltaproteobacteriaOscillatoriophycideaeUnassigned taxonomy

a

40

a

c e

30

be

20Num

ber

of c

lone

s

c

10

a b

cd

e

e

0RSASSS2SSI

b

cd

Soil types

Fig. 1. Taxonomic distribution of different major functional microbial guilds envisaged across four different soil habitats.

Fig. 2. Venn diagrams representing the observed overlap of OTUs for

nifH gene libraries (distance = 0.05) between soils. The values in the

diagram represent the number of genera that were taxonomically

classified.

FEMS Microbiol Lett && (2014) 1–9ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

4 B. Yousuf et al.

the habitats, using PAST software. Five different critical

environmental variables were observed that provide selec-

tive pressure for the bacterial diversity at each habitat.

The CCA plot of soil characteristics, abundances of nifH

genes and bacterial taxonomic groups from four habitats

revealed that axis 1 was positively correlated to salinity

(EC, electrolytic conductivity), pH, TS (total sulphur con-

tent) and TC (total carbon content), whereas axis 2

accounted for the negative correlation between TC and

TN (total nitrogen content) at P < 0.05 (Fig. 3). EC, TS

and pH were positively correlated with saline soils (SS1

and SS2), whereas TC and TN were positively correlated

with agriculture and rhizosphere soils (P < 0.05).

Composite phylogeny

The composite nifH gene phylogeny was inferred based

on the maximum-likelihood method using GTR model

with discrete Gamma distribution, which showed site-spe-

cific as well as a random distribution of phylotypes and

resulted into nine major clusters. The subclusters of clus-

ter 1 and 9 along with cluster 3 possessed saline soil

clones only. Almost 50% of SS1 and SS2 clones did not

clustered to the nifH gene sequences of the characterized

genera. The nifH gene sequences were also randomly dis-

tributed in numerous clades, such as clusters 1–2, 4–7and 9, which showed affiliation to the Alphaproteobacte-

ria, Betaproteobacteria, Deltaproteobacteria, Gammaproteo-

bacteria, Clostridiales and Bacillales groups (Fig. 4). Some

of the phylotypes were tightly clustered to nifH gene

sequences of some recognized genera, such as H. modes-

ticaldum, Chroococcidiopsis thermalis, Ectothiorhodospira

haloalkaliphila, Halorhodospira halophila, Paenibacillus

azotofixans, Sinorhizobium medicae, M. szegediense, Brady-

rhizobium and Desulfovibrio. Cluster 6–8 had agricultural

and rhizosphere soil clones only, of which 30% did not

group with nifH gene sequences of recognized cultured

bacteria (Fig. 4).

Discussion

This study describes the comparative exploration of func-

tional diversity and quantification of nifH genes, epito-

mizing the diazotrophic bacterial biota, associated with

contrasting coastal–saline, agricultural and rhizosphere

soil niches using a gene-targeted clone library approach.

Greater knowledge of the diazotrophic microbial commu-

nities involved in nitrogen transformations is necessary to

understand this globally important complex biogeochemi-

cal cycle, its interlinking with carbon and sulphur bio-

chemical cycling and counteracting nitrogen pollution. In

any ecosystem, nitrogen fixation is highly influenced by

resident functional microbial guilds, plant community

and physicochemical characteristics of the habitat (Santos

et al., 2011).

In our previous studies, we have analysed the compara-

tive community structure across these habitats based on 16S

rRNA and functional genes (Yousuf et al., 2012a, b, 2014).

Here, we envisaged the differential distribution of diazo-

trophs across these habitats by targeting the nifH gene.

Although nifH gene-based diazotrophy is considered to be

widespread and has been recently addressed in many habi-

tats, such as mangrove sediments (Cavalcante et al., 2012),

rhizosphere soil (Chowdhury et al., 2009), soybean field soil

(Xiao et al., 2010) and soil microenvironments (Izquierdo

& N€usslein, 2006), knowledge about the nitrogen cycling

remains scarce in coastal barren–saline soil environments.

The majority of phylotypes from the low saline soil

(SS1) gene library belong to Gammaproteobacteria (45

clones) and Alphaproteobacteria (15), whereas the high

saline soil clone library (SS2) was represented by Alpha-

proteobacteria (59 clones) and Gammaproteobacteria (26)

phylogenetic groups (Fig. 1). This is in accordance with

previous reports, in which dominance of Alphaproteobac-

teria and Gammaproteobacteria was observed in saline/

hypersaline environments (Benlloch et al., 2002; Wu et al.,

2006). Moreover, the dominance of nifH gene allied

Alphaproteobacteria clones was also consistent with a

report from coastal sand (Gobet et al., 2012). However,

Keshri et al. (2013) reported the dominance of Alphapro-

teobacteria and Betaproteobacteria in a saline soil ecosystem

but their results were based on just a few representative

clones.

The Betaproteobacteria allied nifH clones were exclu-

sively present in agriculture and rhizosphere soils and

–3.0 –2.5 –2.0 –1.5 –1.0 –0.5 0.0 0.5 1.0 1.5 2.0–2.4

–1.6

–0.8

0.0

0.8

1.6

2.4

3.2

4.0

4.8

5.6

TC

TN

pHEC TS

BacillalesEubacteria

Betaproteobacteria

Deltaproteobacteria

AlphaproteobacteriaRS

ASSS2

SS1

nifH

OTUs

GammaproteobacteriaUnassignedOscillatoriophycideae

Clostridia

CCA1

CCA2

Pleurocapsales

Fig. 3. CCA showing the distribution of major taxonomic groups

retrieved from saline (SS1 & SS2), agricultural (AS) and rhizosphere

soil (RS) systems along with selected environmental variables of these

four sites.

FEMS Microbiol Lett && (2014) 1–9 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Diazotrophs in coastal saline soil ecosystems 5

absent from saline soils. The Alphaproteobacteria and

Betaproteobacteria are dominant players in root-associated

soil (Wu et al., 2009). The nifH gene sequences affiliated

to Alphaproteobacteria, Betaproteobacteria and Gammapro-

teobacteria taxonomic groups have been reported from

rhizosphere, bulk paddy soil (Shu et al., 2012) and oligo-

trophic tropical seagrass beds (Bagwell et al., 2002). These

reports, along with this study, reinforce the role of Alpha-

proteobacteria and Gammaproteobacteria in coastal–salinesoil ecosystems and Alphaproteobacteria and Betaproteo-

bacteria in agricultural ecosystems, as nitrogen fixers. The

barren–saline soil sites harboured a rich repertoire of

novel nifH gene clones, whereas the majority of agricul-

ture and rhizosphere soil clones were affiliated to well-

known nitrogen-fixing microbial genera.

The nifH phylotypes related to Alphaproteobacteria gen-

era, such as Bradyrhizobium (23), R. radiobacter (11) and

Amorphomonas oryzae (13), were observed at saline soil

sites, emphasizing their contribution in nitrogen cycling

at these barren–saline sites. These genera possibly have

the ability to reside in/adapt to arid and stressful environ-

mental conditions. The high abundance of Bradyrhizobi-

um-affiliated sequences is noteworthy as they are known

to be excellent survivors across diverse conditions and fix

nitrogen in symbiotic association. Peanut preferentially

nodulates with Bradyrhizobium (Yousuf et al., 2012a;

Wang et al., 2013b) and the studied sites (AS and RS)

were regularly cropped with peanuts. The presence of

Bradyrhizobium was also observed at saline soils, which

may be due to the symbiosis between coastal plants and

Bradyrhizobium sp. (Fonseca et al., 2012). The genetic

potential to fix N2 by Cyanobacteria was predominantly

observed at the SS1 site only, which was consistent with

the study of Benlloch et al. (2002). The presence of Delta-

proteobacteria sulphate reducers, such as Desulfovibrio,

Desulfomicrobium and Geoalkalibacter, at saline soils indi-

cates that Deltaproteobacteria play an active role in these

ecosystems. Sulphate-reducing bacteria belonging to order

Desulfovibrionales have been reported to be strongly

adapted to environmental stresses, such as anthropogenic

heavy metal contamination (Quillet et al., 2012) and oil

Fig 4. Composite maximum-likelihood tree constructed from

representative nifH gene sequences. The analysis included 259

nucleotide sequences consisting of sequences from low saline (SS1),

high saline (SS2), agricultural (AS) and rhizosphere soil clone libraries

and closely related nifH gene sequences from known cultured

representatives and environmental clones. Only one representative

sequence from each OTU is used for tree construction. Representative

sequences of the SS1 site (low saline soil) are in green, SS2 site (high

saline soil) in blue, AS site (agriculture soil) in pink and RS site

(rhizosphere soil) in red. The scale bar indicates 0.02 substitutions.

The nifH gene sequence of Methanococcus jannaschii was used as

outgroup for tree calculations.

FEMS Microbiol Lett && (2014) 1–9ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

6 B. Yousuf et al.

contamination (Miralles et al., 2007). The sulphate-reduc-

ing bacteria are biogeochemically important organisms,

especially for the degradation of organic matter in marine

sediments (Blazejak & Schippers, 2011).

Recently, the dominance of Azospirillum, Bradyrhizobi-

um and Rhizobium was observed at four terrestrial cli-

matic zones by targeted metagenomics (Wang et al.,

2013a). Our results corroborate that B. japonicum (AS,

RS; 9, 22 clones), Azospirillum zeae (4, 20) and A. brasil-

iense (15, 4) may be ubiquitously present in agriculture

ecosystems. Azospirillum performs nonsymbiotic nitrogen

fixation and is known to be associated with roots of

grasses, cereals, food crops and soils (Peng et al., 2006).

Additionally, Azohydromonas australica (1, 25 clones),

I. dechloratans (13, 11) and Paenibacillus (AS, 7) -related

phylotypes were also found in high numbers, highlighting

their critical role in these ecosystems. An Azoarcus-related

nifH phylotype was observed in the AS rather than RS

site, and these organisms are known to fix nitrogen as

symbionts within plant roots (Reinhold-Hurek & Hurek,

1997). Results suggest that nitrogen fixation may also be

the main process in addition to carbon fixation in saline

soil ecosystems and that diazotrophy is widespread in

both marine and terrestrial chemosynthetic ecosystems

(Sarbu et al., 1996; Chen et al., 2009; Gray & Engel,

2013).

The average nifH gene copies per gram of SS1 soil was

1.66 9 108 higher than SS2, AS and RS (2.0 9 107, 1.33 9

107 and 7.0 9 106, respectively; SS1 > SS2 > AS > RS;

Table 1). Such differences in copy number may occur

because of variation in the bioavailability of organic car-

bon, sulphur and nitrogen (Andrade et al., 2012; Quillet

et al., 2012). Similarly, the comparable copy numbers were

quantified from the rhizosphere and bulk paddy soil under

different duration of organic management and the abun-

dance was increased with organic management (Shu et al.,

2012). The quantification of nifH gene copy numbers in

the rhizosphere of sorghum has also been performed, and

was somewhat similar to the present data (Coelho et al.,

2008). Higher nifH gene copy numbers were observed in

saline soils (SS1 and SS2) than agriculture and rhizosphere

soils, which is in concordance with Pereira-e-Silva et al.

(2013). The nifH genes have been reported to be present in

good numbers in various terrestrial habitats (Levy-Booth &

Winder, 2010; Xiao et al., 2013). The saline soils are devoid

of vegetation, and therefore lower cycling of organic matter

is expected, which allows diazotrophs to flourish. The

results also suggest that more diverse nifH phylotypes are

expected to be recovered from these sites by additional

sequencing efforts. Nevertheless, this study has still

detected a pattern in the distribution of diazotrophic

microbial guilds across these sites, which in turn will

provide a platform for designing a modified isolation

culture medium, ecological understanding and ascertaining

diazotrophic physiology at these sites.

The present study has advanced our understanding of

the nitrogen cycle in four distinctive environmental

niches and revealed novel lineages of important func-

tional diazotrophic microbial groups, specifically occur-

ring in saline and agriculture/rhizospheric soil ecosystems

(Table S2). Insight into microorganisms in the bulk and

rhizosphere ecosystems is important for the understand-

ing of nutrient cycling processes, ecosystem functioning

and interlinking with community structure. To gain a

better understanding of nitrogen cycling, functional

metagenomic studies should be combined with metatran-

scriptomic/metaproteomic approaches and stable isotope

probing, which will reveal the functional dynamics of a

given community and also provide insight into novel

physiologies and structure–function relationships of resi-

dent microbial communities. Furthermore, we can also

explore the selection of potential salt-tolerant diazotroph-

ic bacterial communities for ameliorating the salt stress in

cropping regimes for sustainable agricultural production.

Acknowledgements

CSIR-CSMCRI Communication No. PRIS-061/2014. The

financial support of CSIR (BSC0117–PMSI and BSC0109–SIMPLE), Government of India, for carrying out this pro-

ject is gratefully acknowledged. B.Y. acknowledges the

Senior Research Fellowship from CSIR, New Delhi, India.

Authors’ contribution

A.M. and B.J. should be considered as joint correspond-

ing authors.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Composite maximum-likelihood tree constructed

from nifH gene sequences to determine variation within

the site.

Fig. S2. Abundance (copy numbers) of the nifH gene

determined by quantitative PCR.

Fig. S3. Rarefaction curves for nifH gene clone libraries at

0.05 cut-off.

Fig. S4. UNIFRAC analysis of nifH gene clone assemblages

from four clone libraries.

Table S1. Physicochemical properties of the soil samples.

Table S2. nifH DNA clones from different soil types

showing similarity with the database analysed by BLAST.

Data S1. Site description and sampling.

FEMS Microbiol Lett && (2014) 1–9 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Diazotrophs in coastal saline soil ecosystems 9