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.     

Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora

N(2) 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 gamma subdivision of the division Proteobacteria (gamma-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the alpha-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.     

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     

Meta-analysis of diazotrophic signatures across terrestrial ecosystems at the continental scale

Biological nitrogen fixation performed by diazotrophs forms a cornerstone of Earth's terrestrial ecosystem productivity. However, the composition, diversity and distribution of soil diazotrophs are poorly understood across different soil ecosystems. Furthermore, the biological potential of the key diazotroph species in relation to key environmental parameters is unknown. To address this, we used meta-analysis approach to merge together 39 independent diazotroph amplicon sequencing (nifH gene) datasets consisting of 1988 independent soil samples. We then employed multiple statistical analyses and machine-learning approaches to compare diazotroph community differences and indicator species between terrestrial ecosystems on a global scale. The distribution, composition and structure of diazotroph communities varied across seven different terrestrial ecosystems, with community composition exhibiting an especially clear effect. The Cyanobacteria were the most abundant taxa in crust ecosystems (accounting for ~45% of diazotrophs), while other terrestrial ecosystems were dominated by Proteobacteria, including Alpha-, Beta- and Gamma-Proteobacteria (accounting for ~70% of diazotrophs). Farmland ecosystems harboured the highest and crust ecosystems the lowest alpha and phylogenetic diversities. Azospirillum zeae, Skermanella aerolata and four Bradyrhizobium species were identified as key indicator species of potential diazotroph activity. Overall, diazotroph abundances and distribution were affected by multiple environmental parameters, including soil pH, nitrogen, organic carbon, C:N ratio and annual mean precipitation and temperature. Together, our findings suggest that based on the relative abundance and diversity of nifH marker gene, diazotrophs have adapted to a range of environmental niches globally.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Response of Free-Living Nitrogen-Fixing Microorganisms to Land Use Change in the Amazon Rainforest

The Amazon rainforest, the largest equatorial forest in the world, is being cleared for pasture and agricultural use at alarming rates. Tropical deforestation is known to cause alterations in microbial communities at taxonomic and phylogenetic levels, but it is unclear whether microbial functional groups are altered. We asked whether free-living nitrogen-fixing microorganisms (diazotrophs) respond to deforestation in the Amazon rainforest, using analysis of the marker gene nifH. Clone libraries were generated from soil samples collected from a primary forest, a 5-year-old pasture originally converted from primary forest, and a secondary forest established after pasture abandonment. Although diazotroph richness did not significantly change among the three plots, diazotroph community composition was altered with forest-to-pasture conversion, and phylogenetic similarity was higher among pasture communities than among those in forests. There was also 10-fold increase in nifH gene abundance following conversion from primary forest to pasture. Three environmental factors were associated with the observed changes: soil acidity, total N concentration, and C/N ratio. Our results suggest a partial restoration to initial levels of abundance and community structure of diazotrophs following pasture abandonment, with primary and secondary forests sharing similar communities. We postulate that the response of diazotrophs to land use change is a direct consequence of changes in plant communities, particularly the higher N demand of pasture plant communities for supporting aboveground plant growth.     

Heat stress reduces the contribution of diazotrophs to coral holobiont nitrogen cycling

Efficient nutrient cycling in the coral-algal symbiosis requires constant but limited nitrogen availability. Coral-associated diazotrophs, i.e., prokaryotes capable of fixing dinitrogen, may thus support productivity in a stable coral-algal symbiosis but could contribute to its breakdown when overstimulated. However, the effects of environmental conditions on diazotroph communities and their interaction with other members of the coral holobiont remain poorly understood. Here we assessed the effects of heat stress on diazotroph diversity and their contribution to holobiont nutrient cycling in the reef-building coral Stylophora pistillata from the central Red Sea. In a stable symbiotic state, we found that nitrogen fixation by coral-associated diazotrophs constitutes a source of nitrogen to the algal symbionts. Heat stress caused an increase in nitrogen fixation concomitant with a change in diazotroph communities. Yet, this additional fixed nitrogen was not assimilated by the coral tissue or the algal symbionts. We conclude that although diazotrophs may support coral holobiont functioning under low nitrogen availability, altered nutrient cycling during heat stress abates the dependence of the coral host and its algal symbionts on diazotroph-derived nitrogen. Consequently, the role of nitrogen fixation in the coral holobiont is strongly dependent on its nutritional status and varies dynamically with environmental conditions.Subject terms: Microbial ecology, Climate-change ecology     

Climate change affects key nitrogen-fixing bacterial populations on coral reefs

Coral reefs are at serious risk due to events associated with global climate change. Elevated ocean temperatures have unpredictable consequences for the ocean''s biogeochemical cycles. The nitrogen cycle is driven by complex microbial transformations, including nitrogen fixation. This study investigated the effects of increased seawater temperature on bacteria able to fix nitrogen (diazotrophs) that live in association with the mussid coral Mussismilia harttii. Consistent increases in diazotroph abundances and diversities were found at increased temperatures. Moreover, gradual shifts in the dominance of particular diazotroph populations occurred as temperature increased, indicating a potential future scenario of climate change. The temperature-sensitive diazotrophs may provide useful bioindicators of the effects of thermal stress on coral reef health, allowing the impact of thermal anomalies to be monitored. In addition, our findings support the development of research on different strategies to improve the fitness of corals during events of thermal stress, such as augmentation with specific diazotrophs.     

Fertilization and defoliation frequency affect genetic diversity of Festuca pratensis Huds. in permanent grasslands

Permanent pastures and meadows are species-rich vegetation systems that play an important role in the ecology and agriculture of temperate climates. Intensive management reduces species diversity and may also influence the genetic diversity within individual species and populations. The objective of this study was to assess genetic variability of meadow fescue, an important component of species-rich grasslands, and to determine whether fertilization and defoliation frequency influence genetic variability within natural populations. Genetic diversity of six natural populations and three cultivars of Festuca pratensis was investigated using randomly amplified polymorphic DNA (RAPD) markers and agronomic traits. Samples of natural populations were taken from two unrelated long-term experiments, where treatments had been applied for 11–38 years. RAPD analysis detected a clear genetic distinction of the cultivars from the natural populations. Genetic variability within cultivars was lower than within natural populations. Analysis of molecular variance ( AMOVA ) showed a significant effect of management on genetic variability. Fertilization and frequent defoliation led to a reduction in genetic variability within natural populations. Analysis of agronomic traits was only partially congruent with the results of RAPD analysis. This study shows that significant genetic variability exists within cultivars and natural populations of Festuca pratensis and can be reduced by intensive management.     

Close Association of Azospirillum and Diazotrophic Rods with Different Root Zones of Kallar Grass

The populations of diazotrophic and nondiazotrophic bacteria were estimated in the endorhizosphere and on the rhizoplane of Kallar grass (Leptochloa fusca) and in nonrhizosphere soil. Microaerophilic diazotrophs were counted by the most-probable-number method, using two semisolid malate media, one of them adapted to the saline-sodic Kallar grass soil. Plate counts of aerobic heterotrophic bacteria were done on nutrient agar. The dominating N₂-fixing bacteria were differentiated by morphological, serological, and physiological criteria. Isolates, which could not be assigned to a known species, were shown to fix nitrogen unequivocally by ¹⁵N₂ incorporation. On the rhizoplane we found 2.0 × 10⁷ diazotrophs per g (dry weight) of root, which consisted in equal numbers of Azospirillum lipoferum and Azospirillum-like bacteria showing characteristics different from those of known Azospirillum species. Surface sterilization by NaOCI treatment effectively reduced the rhizoplane population, so that bacteria released by homogenization of roots could be regarded as endorhizosphere bacteria. Azospirillum spp. were not detected in the endorhizosphere, but diazotrophic, motile, straight rods producing a yellow pigment occurred with 7.3 × 10⁷ cells per g (dry weight) of root in the root interior. In nonrhizosphere soil we found 3.1 × 10⁴ nitrogen-fixing bacteria per g. Diazotrophs were preferentially enriched in the Kallar grass rhizosphere. In nonrhizosphere soil they made up 0.2% of the total aerobic heterotrophic microflora, on the rhizoplane they made up 7.1%, and in the endorhizosphere they made up 85%. Owing to high numbers in and on roots and their preferential enrichment, we concluded that diazotrophs are in close association with Kallar grass. They formed entirely different populations on the rhizoplane and in the endorhizosphere.     

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.     

Impacts of warming and fertilization on nitrogen-fixing microbial communities in the Canadian High Arctic

The impacts of simulated climate change (warming and fertilization treatments) on diazotroph community structure and activity were investigated at Alexandra Fiord, Ellesmere Island, Canada. Open Top Chambers, which increased growing season temperatures by 1-3 degrees C, were randomly placed in a dwarf-shrub and cushion-plant dominated mesic tundra site in 1995. In 2000 and 2001 20N:20P2O5:20K2O fertilizer was applied at a rate of 5 gm(-2) year(-1). Estimates of nitrogen fixation rates were made in the field by acetylene reduction assays (ARA). Higher rates of N fixation were observed 19-35 days post-fertilization but were otherwise unaffected by treatments. However, moss cover was significantly positively associated with ARA rate. NifH gene variants were amplified from bulk soil DNA and analyzed by terminal restriction fragment length polymorphism analysis. Non-metric multidimensional scaling was used to ordinate treatment plots in nifH genotype space. NifH gene communities were more strongly structured by the warming treatment late in the growing season, suggesting that an annual succession in diazotroph community composition occurs.