Molecular Analysis of Diazotroph Diversity in the Rhizosphere of the Smooth Cordgrass, Spartina alterniflora

N₂ fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria (γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.     

Recovery and phylogenetic analysis of nifH sequences from diazotrophic bacteria associated with dead aboveground biomass of Spartina alterniflora.

DNA was extracted from dry standing dead Spartina alterniflora stalks as well as dry Spartina wrack from the North Inlet (South Carolina) and Sapelo Island (Georgia) salt marshes. Partial nifH sequences were PCR amplified, the products were separated by denaturing gradient gel electrophoresis (DGGE), and the prominent DGGE bands were sequenced. Most sequences (109 of 121) clustered with those from alpha-Proteobacteria, and 4 were very similar (>99%) to that of Azospirillum brasilense. Seven sequences clustered with those from known gamma-Proteobacteria and five with those from known anaerobic diazotrophs. The diazotroph assemblages associated with dead Spartina biomass in these two salt marshes were very similar, and relatively few major lineages were represented.     

Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening

Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.     

Responses of Salt Marsh Plant Rhizosphere Diazotroph Assemblages to Changes in Marsh Elevation,Edaphic Conditions and Plant Host Species

An important source of new nitrogen in salt marsh ecosystems is microbial diazotrophy (nitrogen fixation). The diazotroph assemblages associated with the rhizospheres (sediment directly affected by the roots) of salt marsh plants are highly diverse, somewhat stable, and consist mainly of novel organisms. In Crab Haul Creek Basin, North Inlet, SC, the distribution of plant types into discrete zones is dictated by relatively minor differences in marsh elevation and it was hypothesized that the biotic and abiotic properties of the plant zones would also dictate the composition of the rhizosphere diazotroph assemblages. Over a period of 1 year, rhizosphere sediments were collected from monotypic stands of the black needlerush, Juncus roemerianus, the common pickleweed, Salicornia virginica, the short and tall growth forms of the smooth cordgrass Spartina alterniflora, and a mixed zone of co-occurring S. virginica and short form, S. alterniflora. DNA was extracted, purified and nifH sequences PCR amplified for denaturing gradient gel electrophoresis (DGGE) analysis to determine the composition of the diazotroph assemblages. The diazotroph assemblages were strongly influenced by season, abiotic environmental parameters and plant host. Sediment chemistry and nitrogen fixation activity were also significantly influenced by seasonal changes. DGGE bands that significantly affected seasonal and zone specific clustering were identified and most of these sequences were from novel diazotrophs, unaffiliated with any previously described organisms. At least one third of the recovered nifH sequences were from a diverse assemblage of Chlorobia, and γ-, α-, β- and δ-Proteobacteria. Diazotrophs that occurred throughout the growing season and among all zones (frequently detected) were also mostly novel. These significant sequences indicated that diazotrophs driving the structure of the assemblages were diverse, versatile, and some were ubiquitous while others were seasonally responsive. Several ubiquitous sequences were closely related to sequences of actively N₂ fixing diazotrophs previously recovered from this system. These sequences from ubiquitous and versatile organisms likely indicate the diazotrophs in these rhizosphere assemblages that significantly contribute to ecosystem function.     

Persistence of selected Spartina alterniflora rhizoplane diazotrophs exposed to natural and manipulated environmental variability

Rhizoplane-rhizosphere nitrogen-fixing microorganisms (diazotrophs) are thought to provide a major source of biologically available nitrogen in salt marshes dominated by Spartina alterniflora. Compositional and functional stability has been demonstrated for this important functional group; however, the quantitative responses of specific diazotroph populations to environmental variability have not been assessed. Changes in the relative abundances of selected rhizoplane diazotrophs in response to long-term fertilization were monitored quantitatively by reverse sample genome probing. Fertilization stimulated Spartina, with plant height nearly tripling after 1 year. Fertilization also resulted in significant changes in interstitial porewater parameters. Diazotrophic activity (acetylene reduction assay) was sensitive to the fertilization treatments and was inhibited in some plots on several sampling dates. However, inhibition was never consistent across all of the replicates within a treatment and activity always recovered. The rhizoplane diazotrophs were quite responsive to environmental variability and to experimental treatments, but none were displaced by either environmental variability or experimental treatments. All strains were detected consistently throughout this study, and extensive spatial heterogeneity in the distribution patterns of these organisms was observed. The physiological traits that differentiate the diazotroph populations presumably support competitiveness and niche specialization, resulting in the observed resilience of the diazotroph populations in the rhizosphere.     

Recovery and Phylogenetic Analysis of nifH Sequences from Diazotrophic Bacteria Associated with Dead Aboveground Biomass of Spartina alterniflora

DNA was extracted from dry standing dead Spartina alterniflora stalks as well as dry Spartina wrack from the North Inlet (South Carolina) and Sapelo Island (Georgia) salt marshes. Partial nifH sequences were PCR amplified, the products were separated by denaturing gradient gel electrophoresis (DGGE), and the prominent DGGE bands were sequenced. Most sequences (109 of 121) clustered with those from α-Proteobacteria, and 4 were very similar (>99%) to that of Azospirillum brasilense. Seven sequences clustered with those from known γ-Proteobacteria and five with those from known anaerobic diazotrophs. The diazotroph assemblages associated with dead Spartina biomass in these two salt marshes were very similar, and relatively few major lineages were represented.     

Nitrogen-fixing phylotypes of Chesapeake Bay and Neuse River estuary sediments

Sediments often exhibit low rates of nitrogen fixation, despite the presence of elevated concentrations of inorganic nitrogen. The organisms that potentially fix nitrogen in sediments have not previously been identified. Amplification of nifH genes with degenerate primers was used to assess the diversity of diazotrophs in two distinct sediment systems, anoxic muds of Chesapeake Bay and shallow surficial sediments of the Neuse River. Phylogenetic analysis revealed that sequences obtained from mid-Chesapeake Bay, which receive high organic loading and are highly reducing, clustered closely with each other and with known anaerobic microorganisms, suggesting a low abundance of aerobic or facultative diazotrophs in these sediments. Sulfate reduction dominates in the surface, but methanogenesis becomes more important with depth. A thin (<1 cm) oxidized layer is present only in the spring. No archaeal nifH sequences were obtained from Chesapeake Bay. Sequences of nifH amplified from surficial sediments of the Neuse River were distant from Chesapeake Bay sequences and included nif phylotypes related to sequences previously reported from marine mats and the Spartina rhizosphere. Differences in environmental site characteristics appear to select for different types of sediment diazotrophs, which is reflected in the phylogenetic composition of amplified nifH sequences.     

Seasonal Variability of Diazotroph Assemblages Associated with the Rhizosphere of the Salt Marsh Cordgrass, Spartina alterniflora

Nitrogen fixation is the primary N source in the highly productive but N-limited North Inlet, SC, USA salt marsh system. The diverse assemblages of nitrogen-fixing (diazotrophic) bacteria associated with the rhizospheres of the short and tall growth forms of Spartina alterniflora were analyzed at two sites, Crab Haul Creek and Goat Island, which are in different tidal creek drainage systems in this marsh. The sites differed in proximity to the main channel for tidal intrusion and in several edaphic parameters. We hypothesized that either the differing abiotic environmental regimes of the two sites or the variation due to seasonal effects result in differences in the diazotroph assemblage. Rhizosphere samples were collected seasonally during 1999 and 2000. DNA was purified and nifH amplified for denaturing gradient gel electrophoresis (DGGE) analysis of diazotroph assemblage composition. Principal components analysis was used to analyze the binary DGGE band position data. Season strongly influenced assemblage composition and biplots were used to identify bands that significantly affected the seasonal and site-specific clustering. The types of organisms that were most responsive to seasonal or site variability were identified on the basis of DGGE band sequences. Seasonally responsive members of the anaerobic diazotrophs were detected during the winter and postsenescence conditions and may have been responsible for elevated pore water sulfide concentrations. Sequences from a diverse assemblage of Gammaproteobacteria were predominant during growth periods of S. alterniflora. Abiotic environmental parameters strongly influenced both the S. alterniflora and the diazotrophic bacterial assemblages associated with this keystone salt marsh plant species.     

Persistence of Selected Spartina alterniflora Rhizoplane Diazotrophs Exposed to Natural and Manipulated Environmental Variability

Rhizoplane-rhizosphere nitrogen-fixing microorganisms (diazotrophs) are thought to provide a major source of biologically available nitrogen in salt marshes dominated by Spartina alterniflora. Compositional and functional stability has been demonstrated for this important functional group; however, the quantitative responses of specific diazotroph populations to environmental variability have not been assessed. Changes in the relative abundances of selected rhizoplane diazotrophs in response to long-term fertilization were monitored quantitatively by reverse sample genome probing. Fertilization stimulated Spartina, with plant height nearly tripling after 1 year. Fertilization also resulted in significant changes in interstitial porewater parameters. Diazotrophic activity (acetylene reduction assay) was sensitive to the fertilization treatments and was inhibited in some plots on several sampling dates. However, inhibition was never consistent across all of the replicates within a treatment and activity always recovered. The rhizoplane diazotrophs were quite responsive to environmental variability and to experimental treatments, but none were displaced by either environmental variability or experimental treatments. All strains were detected consistently throughout this study, and extensive spatial heterogeneity in the distribution patterns of these organisms was observed. The physiological traits that differentiate the diazotroph populations presumably support competitiveness and niche specialization, resulting in the observed resilience of the diazotroph populations in the rhizosphere.     

Physiological Diversity of the Rhizosphere Diazotroph Assemblages of Selected Salt Marsh Grasses

Rhizosphere diazotroph assemblages of salt marsh grasses are thought to be influenced by host plant species and by a number of porewater geochemical parameters. Several geochemical variables can adversely affect plant productivity and spatial distributions, resulting in strong zonation of plant species and growth forms. This geochemically induced stress may also influence the species compositions and distributions of rhizosphere diazotroph assemblages, but little is currently known about these organisms. The diversity and key physiological features of culturable, O₂-tolerant rhizosphere diazotrophs associated with the tall and short growth forms of Spartina alterniflora and with Juncus roemerianus were examined. A total of 339 gram-negative strains were isolated by a root stab culture approach and morphologically and physiologically characterized by using API and BIOLOG tests. Eighty-six distinct groups composed of physiologically similar strains were identified. Of these groups, 72% were shown to be capable of N₂ fixation through molecular analyses, and a representative strain was chosen from each diazotroph group for further characterization. Cluster and principal-components analysis of BIOLOG data allowed the designation of physiologically distinct strain groupings. Most of these groups were dominated by strains that were not identifiable to species on the basis of API or BIOLOG testing. Representatives of several families including the Enterobacteriaceae, Vibrionaceae, Azotobacteraceae, Spirillaceae, Pseudomonadaceae, and Rhizobiaceae were recovered, as well as strains with no clear taxonomic affiliations. This study identifies numerous potentially important physiological groups of the salt marsh diazotroph assemblage.     

Stability in Natural Bacterial Communities: I. Nutrient Addition Effects on Rhizosphere Diazotroph Assemblage Composition

Abstract The ability of rhizosphere diazotrophs to remain competitive during increased nitrogen availability in situ was tested in a salt marsh grass stand. Nitrogen (16.3 g m^-2) or nitrogen (16.3 g m^-2) and phosphorus (18.0 g m^-2) were added to plots of short form Spartina alterniflora for either 2 weeks or 8 weeks. The diazotroph assemblage composition was monitored via the polymerase chain reaction using nifH specific primers followed by denaturing gradient gel electrophoresis (DGGE) analysis. DGGE profiles from the short-term experiments (2 and 8 weeks) were compared to profiles from control (no additions) and from long-term (>10 y) nutrient addition plots. Nitrogen fixation activity was assayed in each short-term treatment and control plot using an acetylene reduction technique. The control and nutrient addition DGGE profiles were very similar throughout the short-term experiments. One DGGE band that was prominent in the control plots was not found in the long-term nutrient addition plots. Diazotrophy may provide a competitive advantage for some species in this system, as indicated by results from the long-term nutrient amended plots. However, the rhizosphere environment in situ appears to limit the immediate impacts of increased nutrient availability on the diazotroph assemblage composition. Results from the short-term nutrient amended plots showed no measurable effect on the diazotroph assemblage. These results indicate substantial short-term stability of the diazotroph assemblage composition, but the potential for change in the face of long-term changes in nutrient availability.     

Stability in Natural Bacterial Communities: II. Plant Resource Allocation Effects on Rhizosphere Diazotroph Assemblage Composition

Abstract Plant rhizospheres are dynamic environments in which microbes compete for resources, particularly plant-derived organic carbon/energy sources. Altering plant productivity changes the availability of carbon/energy resources to rhizosphere microbiota. This limitation is expected to intensify competition for the remaining carbon supply and could cause the loss of poor competitors from the assemblage. We clipped or shaded plots of the salt marsh cord grass, Spartina alterniflora, to shift plant carbon resource allocation from the rhizosphere to the aboveground shoots. We then examined key porewater parameters (pH, salinity, H2S, NH⁺4), diazotrophic activity (by acetylene reduction assay), and diazotroph assemblage composition after 2 weeks or 8 weeks of treatment. The diazotroph assemblage composition was monitored via the polymerase chain reaction using nifH specific primers followed by denaturing gradient gel electrophoresis (DGGE) analysis. Porewater parameters and acetylene reduction rates did not differ significantly among treatments. The DGGE profiles also were very similar across the control and experimental treatments, indicating that no detectable diazotroph species were displaced from the assemblage. This implies that rhizosphere diazotrophs are able to compete successfully against nondiazotrophs, in spite of the high energy requirements of nitrogen fixation. These results show that the species composition of the diazotroph assemblage in the S. alterniflora rhizosphere is stable in the face of short-duration but potentially high-impact variations in carbon resource availability.     

Phylogenetic diversity of nitrogen-fixing bacteria and the nifH gene from mangrove rhizosphere soil

Nine types of nitrogen-fixing bacterial strains were isolated from 3 rhizosphere soil samples taken from mangrove plants in the Dongzhaigang National Mangrove Nature Reserve of China. Most isolates belonged to Gammaproteobacteria Pseudomonas, showing that these environments constituted favorable niches for such abundant nitrogen-fixing bacteria. New members of the diazotrophs were also found. Using a soil DNA extraction and PCR-cloning-sequencing approach, 135 clones were analyzed by restriction fragment length polymorphism (RFLP) analysis, and 27 unique nifH sequence phylotypes were identified, most of which were closely related to sequences from uncultured bacteria. The diversity of nitrogen-fixing bacteria was assessed by constructing nifH phylogenetic trees from sequences of all isolates and clones in this work, together with related nifH sequences from other mangrove ecosystems in GenBank. The nifH diversity varied among soil samples, with distinct biogeochemical properties within a mangrove ecosystem. When comparing different mangrove ecosystems, the nifH gene sequences from a specific site tended to cluster as individual groups. The results provided interesting data and novel information on our understanding of diazotroph community diversity in the mangrove ecosystems.     

Microdiversity of Culturable Diazotrophs from the Rhizoplanes of the Salt Marsh Grasses Spartina alterniflora and Juncus roemerianus

Abstract Salt marshes dominated by Spartina alterniflora (smooth cordgrass) are among the most productive ecosystems known, despite nitrogen limitation. Rhizoplane/rhizosphere diazotrophy (nitrogen fixation) serves as a significant source of combined nitrogen in these systems. Several recent studies have demonstrated remarkable physiological and phylogenetic macro- and microdiversity within this important functional group of organisms. However, the ecological significance of this diversity is presently unknown. The physiological characteristics of the culturable, oxygen-utilizing fraction of the rhizoplane diazotroph assemblages from Spartina alterniflora and from another salt marsh grass, the black needle rush Juncus roemerianus, were examined in combination with an assessment of the phylogenetic relatedness by whole genome DNA–DNA hybridization. Analysis of substrate utilization data permitted quantitative evaluation of fully cross-hybridizing strain groups and physiological clusters. Phylogenetically related strains, defined by DNA homology ≥90% relative to the positive control, displayed extensive physiological diversity. Seven bootstrap-supported physiological clusters, composed largely of phylogenetically dissimilar strains, showed similar utilization patterns for at least one class of ecologically relevant substrates (carbohydrates, carboxylic acids, or amino acids). These diazotrophs appear to be physiologically adapted for utilization of specific substrates or classes of substrates, lending support to diazotrophic functional redundancy. Microenvironmental heterogeneity is credited for promoting this diversity by selecting for physiologically specialized diazotroph populations to occupy defined niches in situ. One outcome of this physiological diversity is maintenance of a crucial environmental function (nitrogen fixation) over a broad range of environmental conditions. Received: 15 October 1999; Accepted: 28 December 1999; Online Publication: 25 April 2000     

New molecular screening tools for analysis of free-living diazotrophs in soil

Free-living nitrogen-fixing prokaryotes (diazotrophs) are ubiquitous in soil and are phylogenetically and physiologically highly diverse. Molecular methods based on universal PCR detection of the nifH marker gene have been successfully applied to describe diazotroph populations in the environment. However, the use of highly degenerate primers and low-stringency amplification conditions render these methods prone to amplification bias, while less degenerate primer sets will not amplify all nifH genes. We have developed a fixed-primer-site approach with six PCR protocols using less degenerate to nondegenerate primer sets that all amplify the same nifH fragment as a previously published PCR protocol for universal amplification. These protocols target different groups of diazotrophs and allowed for direct comparison of the PCR products by use of restriction fragment length polymorphism fingerprinting. The new protocols were optimized on DNA from 14 reference strains and were subsequently tested with bulk DNA extracts from six soils. These analyses revealed that the new PCR primer sets amplified nifH sequences that were not detected by the universal primer set. Furthermore, they were better suited to distinguish between diazotroph populations in the different soils. Because the novel primer sets were not specific for monophyletic groups of diazotrophs, they do not serve as an identification tool; however, they proved powerful as fingerprinting tools for subsets of soil diazotroph communities.     

Spatial and Temporal Assessment of Diazotroph Assemblage Composition in Vegetated Salt Marsh Sediments Using Denaturing Gradient Gel Electrophoresis Analysis

Abstract Diazotroph assemblage compositions were assessed in rhizosphere sediments from the tall and short form Spartina alterniflora growth zones over an annual cycle. Sediment cores were collected for DNA extraction and nitrogenase (acetylene reduction) activity assays, and porewater samples were analyzed for several chemical parameters in March, June, September, and December 1997. These data were collected to determine if within- or between-zone differences in the diazotroph assemblage composition correlated with differences in key environmental variables or acetylene reduction activity. Acetylene reduction rates differed between zones and within a zone over an annual period. Soluble sulfide concentrations were higher in the short form S. alterniflora zone on all dates except those in June and differed within both zones on different sample dates. nifH sequences were recovered from rhizosphere sediment DNA by PCR amplification using nifH specific primers. These amplimers were analyzed using denaturing gradient gel electrophoresis (DGGE), and the resulting patterns were compared by neural network and linear discriminant analyses. Ten prominent amplimers, four of which were apparent heteroduplexes, were observed. DGGE banding profiles showed minor differences among sampling dates and between sample zones, but the overall banding pattern was remarkably consistent. This reflects overall similarity between the amplifiable diazotroph assemblages in the tall and short S. alterniflora growth zones and substantial seasonal stability in assemblage composition. Received: 2 March 1999; Accepted: 4 May 1999     

A global census of nitrogenase diversity

The global diversity of nitrogen-fixing microorganisms was assessed through construction and analysis of an aligned database of 16,989 nifH sequences. We conclude that the diversity of diazotrophs is still poorly described and that many organisms remain to be discovered. Our analyses indicate that diversity is not distributed evenly across phylogenetic groups or across environments and that some of the most diverse assemblages and environments remain the most poorly characterized. The majority of OTUs were rare, falling in the long tail of the frequency distribution. The most dominant OTUs fell into either the Cyanobacteria or the α, β, and γ Proteobacteria, and five of these dominant OTUs do not have any representatives cultivated in isolation. Soils contained the greatest diversity of nifH sequences of all of the environments surveyed. Cluster III, which is dominated by nifH sequences from obligate anaerobes, was found to contain the greatest diversity of all nifH lineages and is also the group for which diversity is the least sampled. Our findings provide context for ongoing efforts to explore diazotroph diversity, indicating specific groups and environments that remain poorly characterized.     

Physiological Diversity of Rhizoplane Diazotrophs of the Saltmeadow Cordgrass,Spartina patens: Implications for Host Specific Ecotypes

Diazotrophic bacteria are important contributors to salt marsh productivity, but the biotic and abiotic factors that influence their distributions and function and the extent of their diversity cannot be understood in the absence of physiological information. Here we examine the physiological diversity and distribution patterns of diazotrophic bacteria associated with the rhizoplane of the saltmeadow cordgrass, Spartina patens, in comparison with diazotrophs from other intertidal grasses (tall and short form Spartina alterniflora and Juncus roemerianus) from the same salt marsh. S. patens plants were collected from two distinct habitats, and a total of 115 strains (111 Gram negative and 4 Gram positive strains) were isolated into pure culture by stab inoculating roots and rhizomes into combined nitrogen-free semisolid media. Most strains were microaerophilic and approximately one-half were motile. API test strips were used to eliminate redundancy within the culture collection, resulting in 21 physiologically different API groups (17 Gram negative and 4 Gram positive groups). A representative strain from each API group was selected for dot blot hybridization with a nifH specific probe and 16 strains (13 Gram negative and 3 Gram positive) were scored as positive. The nifH positive API group representative strains were characterized further using BIOLOG test plates. Substrate utilization potentials defined two S. patens strain clusters, and only one S. patens strain was physiologically similar to any other strain from a different host plant origin. No distinctions could be made based on the different S. patens habitats, suggesting that the host plant may have a greater impact than abiotic environmental conditions on the distributions of the rhizoplane diazotrophs recovered.     

Diversity of free-living nitrogen-fixing microorganisms in the rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings

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 presence of 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 plant communities were collected from two wastelands with different discarded periods. The nested PCR method was used to amplify the nifH genes from environmental DNA extracted from tailing samples. Twenty-two of 37 nifH gene sequences retrieved from DGGE gels clustered in Proteobacteria (α-Proteobacteria and β-Proteobacteria) and 15 nifH gene sequences in Cyanobacteria. Most nifH gene fragments sequenced were closely related to uncultured bacteria and cyanobacteria and exhibited less than 90% nucleotide acid identity with bacteria in the database, suggesting that the nifH gene fragments detected in copper mine tailings may represent novel sequences of nitrogen-fixers. Our results indicated that the non-rhizosphere tailings generally presented higher diversity of nitrogen-fixers than rhizosphere tailings and the diversity of free-living nitrogen-fixers in tailing samples was mainly affected by the physico-chemical properties of the wastelands and plant species, especially the changes of nutrient and heavy metal contents caused by the colonization of plant community.