Accepted Article
Received Date : 11-Jul-2014 Revised Date : 27-Aug-2014 Accepted Date : 31-Aug-2014 Article type
: Research Letter
Editor
: Tim Daniell
Differential distribution and abundance of diazotrophic bacterial communes across different soil niches using gene targeted clone library approach Basit Yousuf 1,2, Raghawendra Kumar 1,2, Avinash Mishra 1,2,* and Bhavanath Jha1,2,* 1
Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals
Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar, Gujarat, India (Pin- 364002)
2
Academy of Scientific and Innovative Research (AcSIR), CSIR, New Delhi
*Corresponding authors: Tel: +91-278-2567760 Ext. 6260; Fax: +91 278 2570885 E-mail addresses: [email protected] (AM) and [email protected] (BJ)
Key Words: Diazotrophs; clone library; microbial diversity; nifH; saline soil
Running title Diazotrophs in coastal saline soil ecosystems
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12593 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Diazotrophs are key players of globally important biogeochemical nitrogen cycle having 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 conditions and the implicated organisms are largely obscure. Differential occurrence of diazotrophs was studied by the nifH gene targeted clone library approach. Four nifH gene clone libraries were constructed from different soil niches i.e. saline soils (low and high saline; EC- 3.8 and 7.1 ds m-1), agricultural and rhizosphere soil. Additionally, abundance of diazotrophic community members was assessed using qPCR. Results showed environment dependent metabolic versatility and presence of nitrogen-fixing bacteria affiliated with a range of taxa, encompassing members of the Alpha-, Beta-, Delta-, Gammaproteobacteria, Cyanobacteria and Firmicutes. The analyses unveiled the dominance of Alpha- and Gammaproteobacteria (Pseudomonas, Halorhodospira, Ectothiorhodospira, Bradyrhizobium, Agrobacterium, Amorphomonas) as nitrogen fixers in coastal-saline soil ecosystems, whereas Alpha- and Betaproteobacteria (Bradyrhizobium, Azohydromonas, Azospirillum, Ideonella) in agricultural/rhizosphere ecosystem. Results revealed a repertoire of novel nitrogen-fixing bacterial guilds particularly at saline soil ecosystems.
Introduction The recycling of nitrogen contributes substantially in nutrient fluxing and sustainable soil fertility in the terrestrial ecosystem (Hsu & Buckley, 2009; Cavalcante et al., 2012). Around half of the annual nitrogen is fluxed into biosphere (Vitousek et al., 1997), natural (Cleveland et al., 1999) and agricultural ecosystems (Peoples et al., 1992) by biological nitrogen fixation, which involves conversion of N2 into ammonia (NH3). This complex process is catalysed by the nitrogenase reductase enzyme, mediated through certain groups of This article is protected by copyright. All rights reserved.
Accepted Article
bacteria/archaea (diazotrophs) in symbiotic, 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 co-ordinately for the functioning of active enzyme (Masepohl et al., 2002; Lee et al., 2000). The nifH gene is widely distributed among phylogenetically diverse bacteria and archaea (Poly et al., 2001a; Dixon & Kahn, 2004). The gene is commonly used for the study of phylogeny, diversity and abundance of both cultured and uncultivated organisms from multiple environments (Zehr et al., 2003), as its protein sequence is well conserved compared to other genes of the nif operon. The relationship among bacteria based on the sequence divergences of this gene has been reported to be in congruent with the 16S rRNA gene phylogeny with some exceptions (Borneman et al., 1996; Ueda et al., 1995; 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; Pereira-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, carbon, nitrogen and sulphur contents has been reported to influence nitrogen fixation in soils (Hsu & Buckley, 2009). A few culture independent studies have been performed on functional diversity of nitrogen-fixing microbial communities in moderate, extreme, terrestrial, bulk and rhizosphere soil environments, such as rice, forest, grasses, soybean and sediments (Hirano et al., 2001, Xiao et al., 2010; Cavalcante et al., 2012; Chowdhury et al., 2009; Orr et al., 2011; 2012), but has not been fairly addressed from saline soil ecosystems (Keshri et al., 2013). Keshri et This article is protected by copyright. All rights reserved.
Accepted Article
al. (2013) focused on only one saline soil and also reported fewer clones (51) and OTUs (20). Saline soil ecosystems represent intriguing ecological niches, where anaerobic or microaerophilic conditions are expected to be prevailing. 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 enzyme of 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 present work was to broaden our view on the diversity and abundance of nitrogen-fixing bacterial communities and their comparative distribution among these environmental niches. Materials and Methods Soil samples and physicochemical characteristics Four distinct sites, comprising of three bulk soil types viz. 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 (Supplementary Text 1). Physicochemical characteristics (Supplementary Table 1) 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 (5′TGCGAYCCSAARGCBGACTC-3′ and 5′-ATSGCCATCATYTCRCCGGA-3′; Poly et al., 2001a). The nifH gene amplicons were purified, cloned in pGEM-T Easy vector, screened for
This article is protected by copyright. All rights reserved.
Accepted Article
correct insert size (360 bp) and positive clones were sequenced (M/s Macrogen Inc., S. 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 put forth to BLASTn program and the most similar 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 operational taxonomic units (OTUs: phylotypes) using Mothur program (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 lowest Bayesian Information Criterion (Tamura et al., 2011). The evolutionary history of all genes was inferred by Maximum Likelihood, using bootstrap resampling method with 500 bootstrap replications. Phylogenetic comparison and statistical analysis Sequence similarity cut-off of 95% (Jiang et al., 2009) was used to define an OTU (phylotype) using Mothur (Schloss et al., 2009). The Jukes-Cantor evolutionary distance matrices were calculated by DNADIST program within the PHYLIP ver. 3.2 (Felsenstein, 1989). The α-diversity indices (ACE and Chao), OTUs, Rarefaction curves, Shannon & Simpson diversity indices and coverage were evaluated using Mothur (Schloss et al., 2009).The datasets were also compared for β-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 environment. The interrelationship between environmental parameters, diversity indices and distribution of
This article is protected by copyright. All rights reserved.
Accepted Article
taxonomic groups was analysed by canonical correspondence analysis (CCA) using PAST version 2.14 (Hammer et al., 2001). Quantitative PCR (qPCR) Absolute quantification of diazotrophic bacteria in each sample was carried out using QuantiFast Kit (Qiagen, USA) with primer pair PolF and PolR, using following PCR program: 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s. The experiments were repeated thrice, independently and the amplified product was run on 1.5% agarose gel to confirm the expected size. The efficiency of qPCR was calculated, data analysed by comparative CT method and copy number of 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 (Supplementary Fig. S1). Clone libraries showed 92-96% similarity with each other and thus indicating a very low variation within the site. The results were supported by weighted UniFrac environmental clustering 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 were 0.9, 1.0, 0.5 and 0.9 respectively). 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 nifH gene was low in RS (0.26 OTUs/ clone), but high in SS1, SS2 and AS (0.40-0.42 OTUs/ clone). The library SS1 revealed the dominance of sequences, affiliated with nifH genes from Gammaproteobacteria (42 clones) followed by Alphaproteobacteria (16) and Deltaproteobacteria (4) phylogenetic groups (Fig.
This article is protected by copyright. All rights reserved.
Accepted Article
1). The different dominant genera encompassed by Proteobacteria include Pseudomonas (15 phylotypes), Halorhodospira halophila (13), filamentous Cyanobacteria (11), Bradyrhizobium japonicum (8) and Ectothiorhodospira (7). Other genera represented by few phylotypes, such as Heliobacterium modesticaldum (5), different species of Azospirillum (5), Amorphomonas oryzae (4), Methylogaea (2), Agrobacterium (3), Ideonella (1), Desulfovibrio (2) and Desulfomicrobium (1). The SS2 clone library had high abundance of Alphaproteobacteria (59 clones) followed by Gammaproteobacteria (26), whereas the most abundant phylotypes that showed affiliation to cultured representatives were Bradyrhizobium (15 clones), Rhizobium radiobacter (11), Amorphomonas oryzae (13) and Halorhodospira halophila (12) (Fig. 1). The library also contained phylotypes related to other genera like Geoalkalibacter (2). These libraries sheltered large pool of novel nifH gene sequences, which showed low similarity to the nifH gene harbouring cultured bacteria and clone sequences from natural environments (Supplementary Table 2). The agricultural and rhizosphere soils were predominantly represented by nifH gene sequences affiliated to Alphaproteobacteria (AS, RS; 47, 58 clones) and Betaproteobacteria (16, 37) (Fig. 1). The highly abundant nifH OTUs showed affiliation with Bradyrhizobium 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), Methylocaldum szegediense (RS-3), Rhizobium radiobacter (4, 2), Pseudomonas (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 sequences of GenBank, however, only 10 and 20% OTUs of SS1 and SS2 clone libraries, respectively showed high nucleotide identity (94-97%). In AS and RS clone libraries, the Gammaproteobacteria, Deltaproteobacteria and Firmicutes phylogenetic groups were represented by few clones. It was observed that Gammaproteobacteria were dominated in This article is protected by copyright. All rights reserved.
Accepted Article
saline soil ecosystems (P
Received Date : 11-Jul-2014 Revised Date : 27-Aug-2014 Accepted Date : 31-Aug-2014 Article type
: Research Letter
Editor
: Tim Daniell
Differential distribution and abundance of diazotrophic bacterial communes across different soil niches using gene targeted clone library approach Basit Yousuf 1,2, Raghawendra Kumar 1,2, Avinash Mishra 1,2,* and Bhavanath Jha1,2,* 1
Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals
Research Institute (CSIR-CSMCRI), G. B. Marg, Bhavnagar, Gujarat, India (Pin- 364002)
2
Academy of Scientific and Innovative Research (AcSIR), CSIR, New Delhi
*Corresponding authors: Tel: +91-278-2567760 Ext. 6260; Fax: +91 278 2570885 E-mail addresses: [email protected] (AM) and [email protected] (BJ)
Key Words: Diazotrophs; clone library; microbial diversity; nifH; saline soil
Running title Diazotrophs in coastal saline soil ecosystems
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1574-6968.12593 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Diazotrophs are key players of globally important biogeochemical nitrogen cycle having 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 conditions and the implicated organisms are largely obscure. Differential occurrence of diazotrophs was studied by the nifH gene targeted clone library approach. Four nifH gene clone libraries were constructed from different soil niches i.e. saline soils (low and high saline; EC- 3.8 and 7.1 ds m-1), agricultural and rhizosphere soil. Additionally, abundance of diazotrophic community members was assessed using qPCR. Results showed environment dependent metabolic versatility and presence of nitrogen-fixing bacteria affiliated with a range of taxa, encompassing members of the Alpha-, Beta-, Delta-, Gammaproteobacteria, Cyanobacteria and Firmicutes. The analyses unveiled the dominance of Alpha- and Gammaproteobacteria (Pseudomonas, Halorhodospira, Ectothiorhodospira, Bradyrhizobium, Agrobacterium, Amorphomonas) as nitrogen fixers in coastal-saline soil ecosystems, whereas Alpha- and Betaproteobacteria (Bradyrhizobium, Azohydromonas, Azospirillum, Ideonella) in agricultural/rhizosphere ecosystem. Results revealed a repertoire of novel nitrogen-fixing bacterial guilds particularly at saline soil ecosystems.
Introduction The recycling of nitrogen contributes substantially in nutrient fluxing and sustainable soil fertility in the terrestrial ecosystem (Hsu & Buckley, 2009; Cavalcante et al., 2012). Around half of the annual nitrogen is fluxed into biosphere (Vitousek et al., 1997), natural (Cleveland et al., 1999) and agricultural ecosystems (Peoples et al., 1992) by biological nitrogen fixation, which involves conversion of N2 into ammonia (NH3). This complex process is catalysed by the nitrogenase reductase enzyme, mediated through certain groups of This article is protected by copyright. All rights reserved.
Accepted Article
bacteria/archaea (diazotrophs) in symbiotic, 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 co-ordinately for the functioning of active enzyme (Masepohl et al., 2002; Lee et al., 2000). The nifH gene is widely distributed among phylogenetically diverse bacteria and archaea (Poly et al., 2001a; Dixon & Kahn, 2004). The gene is commonly used for the study of phylogeny, diversity and abundance of both cultured and uncultivated organisms from multiple environments (Zehr et al., 2003), as its protein sequence is well conserved compared to other genes of the nif operon. The relationship among bacteria based on the sequence divergences of this gene has been reported to be in congruent with the 16S rRNA gene phylogeny with some exceptions (Borneman et al., 1996; Ueda et al., 1995; 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; Pereira-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, carbon, nitrogen and sulphur contents has been reported to influence nitrogen fixation in soils (Hsu & Buckley, 2009). A few culture independent studies have been performed on functional diversity of nitrogen-fixing microbial communities in moderate, extreme, terrestrial, bulk and rhizosphere soil environments, such as rice, forest, grasses, soybean and sediments (Hirano et al., 2001, Xiao et al., 2010; Cavalcante et al., 2012; Chowdhury et al., 2009; Orr et al., 2011; 2012), but has not been fairly addressed from saline soil ecosystems (Keshri et al., 2013). Keshri et This article is protected by copyright. All rights reserved.
Accepted Article
al. (2013) focused on only one saline soil and also reported fewer clones (51) and OTUs (20). Saline soil ecosystems represent intriguing ecological niches, where anaerobic or microaerophilic conditions are expected to be prevailing. 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 enzyme of 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 present work was to broaden our view on the diversity and abundance of nitrogen-fixing bacterial communities and their comparative distribution among these environmental niches. Materials and Methods Soil samples and physicochemical characteristics Four distinct sites, comprising of three bulk soil types viz. 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 (Supplementary Text 1). Physicochemical characteristics (Supplementary Table 1) 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 (5′TGCGAYCCSAARGCBGACTC-3′ and 5′-ATSGCCATCATYTCRCCGGA-3′; Poly et al., 2001a). The nifH gene amplicons were purified, cloned in pGEM-T Easy vector, screened for
This article is protected by copyright. All rights reserved.
Accepted Article
correct insert size (360 bp) and positive clones were sequenced (M/s Macrogen Inc., S. 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 put forth to BLASTn program and the most similar 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 operational taxonomic units (OTUs: phylotypes) using Mothur program (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 lowest Bayesian Information Criterion (Tamura et al., 2011). The evolutionary history of all genes was inferred by Maximum Likelihood, using bootstrap resampling method with 500 bootstrap replications. Phylogenetic comparison and statistical analysis Sequence similarity cut-off of 95% (Jiang et al., 2009) was used to define an OTU (phylotype) using Mothur (Schloss et al., 2009). The Jukes-Cantor evolutionary distance matrices were calculated by DNADIST program within the PHYLIP ver. 3.2 (Felsenstein, 1989). The α-diversity indices (ACE and Chao), OTUs, Rarefaction curves, Shannon & Simpson diversity indices and coverage were evaluated using Mothur (Schloss et al., 2009).The datasets were also compared for β-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 environment. The interrelationship between environmental parameters, diversity indices and distribution of
This article is protected by copyright. All rights reserved.
Accepted Article
taxonomic groups was analysed by canonical correspondence analysis (CCA) using PAST version 2.14 (Hammer et al., 2001). Quantitative PCR (qPCR) Absolute quantification of diazotrophic bacteria in each sample was carried out using QuantiFast Kit (Qiagen, USA) with primer pair PolF and PolR, using following PCR program: 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s. The experiments were repeated thrice, independently and the amplified product was run on 1.5% agarose gel to confirm the expected size. The efficiency of qPCR was calculated, data analysed by comparative CT method and copy number of 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 (Supplementary Fig. S1). Clone libraries showed 92-96% similarity with each other and thus indicating a very low variation within the site. The results were supported by weighted UniFrac environmental clustering 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 were 0.9, 1.0, 0.5 and 0.9 respectively). 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 nifH gene was low in RS (0.26 OTUs/ clone), but high in SS1, SS2 and AS (0.40-0.42 OTUs/ clone). The library SS1 revealed the dominance of sequences, affiliated with nifH genes from Gammaproteobacteria (42 clones) followed by Alphaproteobacteria (16) and Deltaproteobacteria (4) phylogenetic groups (Fig.
This article is protected by copyright. All rights reserved.
Accepted Article
1). The different dominant genera encompassed by Proteobacteria include Pseudomonas (15 phylotypes), Halorhodospira halophila (13), filamentous Cyanobacteria (11), Bradyrhizobium japonicum (8) and Ectothiorhodospira (7). Other genera represented by few phylotypes, such as Heliobacterium modesticaldum (5), different species of Azospirillum (5), Amorphomonas oryzae (4), Methylogaea (2), Agrobacterium (3), Ideonella (1), Desulfovibrio (2) and Desulfomicrobium (1). The SS2 clone library had high abundance of Alphaproteobacteria (59 clones) followed by Gammaproteobacteria (26), whereas the most abundant phylotypes that showed affiliation to cultured representatives were Bradyrhizobium (15 clones), Rhizobium radiobacter (11), Amorphomonas oryzae (13) and Halorhodospira halophila (12) (Fig. 1). The library also contained phylotypes related to other genera like Geoalkalibacter (2). These libraries sheltered large pool of novel nifH gene sequences, which showed low similarity to the nifH gene harbouring cultured bacteria and clone sequences from natural environments (Supplementary Table 2). The agricultural and rhizosphere soils were predominantly represented by nifH gene sequences affiliated to Alphaproteobacteria (AS, RS; 47, 58 clones) and Betaproteobacteria (16, 37) (Fig. 1). The highly abundant nifH OTUs showed affiliation with Bradyrhizobium 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), Methylocaldum szegediense (RS-3), Rhizobium radiobacter (4, 2), Pseudomonas (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 sequences of GenBank, however, only 10 and 20% OTUs of SS1 and SS2 clone libraries, respectively showed high nucleotide identity (94-97%). In AS and RS clone libraries, the Gammaproteobacteria, Deltaproteobacteria and Firmicutes phylogenetic groups were represented by few clones. It was observed that Gammaproteobacteria were dominated in This article is protected by copyright. All rights reserved.
Accepted Article
saline soil ecosystems (P
