Diazotrophic Community Structure Research Articles

Fungal endophyte Phomopsis liquidambari affects nitrogen transformation processes and related microorganisms in the rice rhizosphere.

The endophytic fungus Phomopsis liquidambari performs an important ecosystem service by assisting its host with acquiring soil nitrogen (N), but little is known regarding how this fungus influences soil N nutrient properties and microbial communities. In this study, we investigated the impact of P. liquidambari on N dynamics, the abundance and composition of N cycling genes in rhizosphere soil treated with three levels of N (urea). Ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and diazotrophs were assayed using quantitative real-time polymerase chain reaction and denaturing gradient gel electrophoresis at four rice growing stages (S0: before planting, S1: tillering stage, S2: grain filling stage, and S3: ripening stage). A significant increase in the available nitrate and ammonium contents was found in the rhizosphere soil of endophyte-infected rice under low N conditions. Moreover, P. liquidambari significantly increased the potential nitrification rates, affected the abundance and community structure of AOA, AOB, and diazotrophs under low N conditions in the S1 and S2 stages. The root exudates were determined due to their important role in rhizosphere interactions. P. liquidambari colonization altered the exudation of organic compounds by rice roots and P. liquidambari increased the concentration of soluble saccharides, total free amino acids and organic acids in root exudates. Plant-soil feedback mechanisms may be mediated by the rice-endophyte interaction, especially in nutrient-limited soil.

The Diversity and Co-occurrence Patterns of N₂-Fixing Communities in a CO₂-Enriched Grassland Ecosystem.

Diazotrophs are the major organismal group responsible for atmospheric nitrogen (N2) fixation in natural ecosystems. The extensive diversity and structure of N2-fixing communities in grassland ecosystems and their responses to increasing atmospheric CO2 remain to be further explored. Through pyrosequencing of nifH gene amplicons and extraction of nifH genes from shotgun metagenomes, coupled with co-occurrence ecological network analysis approaches, we comprehensively analyzed the diazotrophic community in a grassland ecosystem exposed to elevated CO2 (eCO2) for 12 years. Long-term eCO2 increased the abundance of nifH genes but did not change the overall nifH diversity and diazotrophic community structure. Taxonomic and phylogenetic analysis of amplified nifH sequences suggested a high diversity of nifH genes in the soil ecosystem, the majority belonging to nifH clusters I and II. Co-occurrence ecological network analysis identified different co-occurrence patterns for different groups of diazotrophs, such as Azospirillum/Actinobacteria, Mesorhizobium/Conexibacter, and Bradyrhizobium/Acidobacteria. This indicated a potential attraction of non-N2-fixers by diazotrophs in the soil ecosystem. Interestingly, more complex co-occurrence patterns were found for free-living diazotrophs than commonly known symbiotic diazotrophs, which is consistent with the physical isolation nature of symbiotic diazotrophs from the environment by root nodules. The study provides novel insights into our understanding of the microbial ecology of soil diazotrophs in natural ecosystems.

Diazotrophic bacterial community variability in a subtropical deep reservoir is correlated with seasonal changes in nitrogen.

Nitrogen-fixing microorganisms (diazotrophs) play important roles in aquatic biogeochemistry and ecosystem functioning. However, little is known about the spatiotemporal variation of diazotrophic microbial communities in deep subtropical reservoirs. In this study, denaturing gradient gel electrophoresis (DGGE), clone libraries, quantitative PCR, and quantitative reverse transcription (RT)-PCR were used together to examine the vertical and seasonal patterns of diazotrophic microbial communities based on nitrogenase (nifH) gene sequences in the Dongzhen Reservoir, China, across time (every 3 months for 1 year) and space (five different water depths). In general, the numbers of DGGE bands increased with water depth during the stratification seasons (spring, summer, and autumn), with the clone-library-based operational taxonomic unit (OTU) number and nifH gene diversity being highest in autumn (6 OTUs at depth 0 m; 15 OTUs at 33 m) and winter (12 OTUs at 0 m, 13 OTUs at 33 m) but decreasing drastically in spring (2 OTUs at 0 m, 3 OTUs at 33 m) and summer (3 OTUs at 0 m, 2 OTUs at 33 m). The nifH gene abundance was lowest in the water mixing season (winter average, 5.17 × 10(7) copies/L) but increased in the three other seasons (9.03 × 10(9) copies/L). Cyanobacteria (dominated by filamentous thermophilic cyanobacteria and Cylindrospermopsis raciborskii) were the most dominant diazotrophic group at all depths and seasons, while both alphaproteobacteria and gammaproteobacteria were co-dominant in the bottom waters in autumn and winter. The distinct seasonal and spatial patterns in diazotrophic communities were significantly related to total nitrogen (TN) and ammonium nitrogen (NH4-N) in the reservoir (P < 0.01). Further, TN showed a significant positive correlation with nifH RNA copy number (P < 0.05) and DGGE band number (P < 0.01), whereas the NH4-N was negatively correlated with nifH DNA copy number (P < 0.01) and positively with both RNA/DNA ratio (P < 0.01) and DGGE band number (P < 0.01). Our data indicated that water stratification, mixing, and nitrogen might drive the diazotrophic community structure and activity in complex ways, thereby influencing the aquatic nitrogen cycle. Therefore, adaptive reservoir management strategies should carefully consider the effects of water stratification for protecting drinking water quality and for controlling the potential for diazotrophic cyanobacteria blooms.

Modeling long‐term change of planktonic ecosystems in the northern South China Sea and the upstream Kuroshio Current

AbstractField studies suggested that the biogeochemical settings and community structures are substantial different between the central Northern South China Sea (NSCS) and the upstream Kuroshio Current (KC). In particular, the water column of KC is characterized by substantially lower nutrients and productivity but higher Trichodesmium abundance and nitrogen fixation compared to the NSCS. The mechanism driving the difference of the two marine ecosystems, however, remains inadequately understood. Here, a one‐dimensional biogeochemical model was developed to simulate the long‐term variability of lower‐trophic planktonic ecosystem for two pelagic stations in the NSCS and the KC near the Luzon Strait. The physical model included the vertical mixing driven by air‐sea interaction and the Ekman pumping induced by wind stress curl. The biological model was constructed by modifying a nitrogen‐based NPZD model with the incorporation of phosphorus cycle and diazotroph nitrogen fixation. After validation by several field data sets, the model was used to study the impact of long‐term physical forcing on ecosystem variability in the two distinct stations. Our results suggested that nutrient transport above nitracline during summer was largely controlled by vertical turbulent mixing, while Ekman pumping was important for nutrient transport below the nitracline. Our results also indicated that diazotroph community structure and N2 fixation in the NSCS and the KC could be strongly influenced by physical processes through the impacts on vertical nutrient fluxes. The disadvantage of diazotroph in the NSCS in compared to the KC during the summer could be attributed to its high nitrate fluxes from subsurface leading to outcompete of diazotrophs by faster growing nondiazotroph phytoplankton.

Nitrogen fixation in Sphagnum mosses is affected by moss species and water table level

Nitrogen (N2) fixation by moss-associated bacteria is an important N source in boreal peatlands and forests. Here we studied whether moss species, water table fluctuations, methane (CH4) availability and diazotroph community structure would affect the rate of Sphagnum-associated N2 fixation. Diazotrophy and methanotrophy were studied in parallel in a double labeling (15 N2 and 13CH4) experiment in forest and fen habitats. The role of N2-fixing methanotrophs was further characterized by the phylogenetic analysis of nifH genes encoding for dinitrogenase reductase. N2 fixation rates were dependent on the moss species in a habitat level, but independent of the diazotroph community structure. Only 6 % of the nifH sequences were taxonomically assigned to cyanobacteria, while the majority (82 %) of genes were assigned to Alphaproteobacteria clustering with the nifH sequences of the order Rhizobiales, but without close matches to sequences of cultivated species. In the originally submerged fen mosses, water increased both N2 fixation and CH4 oxidation rates. However, such effect was not seen in forest mosses grown above the water table level. CH4 addition did not enhance N2 fixation. N2 fixation associated to Sphagnum mosses was controlled by the moss species and the water environment. Although most of the observed nifH sequences were related to the order Rhizobiales, methanotrophs were not responsible for the N2 fixation.

Variation in diazotrophic community structure in forest soils reflects land use history

Microbial nitrogen fixation is an important source of nitrogen in soils of both old and secondary-growth forests. Considering that many soils, which today support secondary-growth forests were once under cultivation, no studies have examined how this kind of disturbance history affects contemporary populations of nitrogen-fixing microbes in forest soils. In the work presented here, we compare secondary-growth forest sites, which were under cultivation more than 150 years ago, to old-growth forest sites in Cadwell Forest, Massachusetts. For each site, nitrogenase activity was measured and the diversity of the nifH gene pools was examined. Three sites with prior agricultural history exhibited higher nitrogenase activity and were dominated by diazotrophs closely related to the α- and γ-Proteobacteria. In contrast, lower nitrogenase activity and the dominance of the anaerobic Cluster III diazotrophs characterized the three old forest sites. Further analyses of species overlap among all six sites revealed that the diazotrophic composition was closely related to previous management history, with agricultural sites clustering together and separate from old forest sites, independent of the proximity between sites. By specifically targeting one of the main functions of microbial communities in soils, the activity and diversity of nitrogen-fixing microorganisms, this work points to a long-lasting effect of former agricultural activities on secondary-growth forest soils, more than one hundred years after succession.

Heterotrophic bacteria as major nitrogen fixers in the euphotic zone of the Indian Ocean

AbstractDiazotrophy in the Indian Ocean is poorly understood compared to that in the Atlantic and Pacific Oceans. We first examined the basin‐scale community structure of diazotrophs and their nitrogen fixation activity within the euphotic zone during the northeast monsoon period along about 69°E from 17°N to 20°S in the oligotrophic Indian Ocean, where a shallow nitracline (49–59 m) prevailed widely and the sea surface temperature (SST) was above 25°C. Phosphate was detectable at the surface throughout the study area. The dissolved iron concentration and the ratio of iron to nitrate + nitrite at the surface were significantly higher in the Arabian Sea than in the equatorial and southern Indian Ocean. Nitrogen fixation in the Arabian Sea (24.6–47.1 μmolN m−2 d−1) was also significantly greater than that in the equatorial and southern Indian Ocean (6.27–16.6 μmolN m−2 d−1), indicating that iron could control diazotrophy in the Indian Ocean. Phylogenetic analysis of nifH showed that most diazotrophs belonged to the Proteobacteria and that cyanobacterial diazotrophs were absent in the study area except in the Arabian Sea. Furthermore, nitrogen fixation was not associated with light intensity throughout the study area. These results are consistent with nitrogen fixation in the Indian Ocean, being largely performed by heterotrophic bacteria and not by cyanobacteria. The low cyanobacterial diazotrophy was attributed to the shallow nitracline, which is rarely observed in the Pacific and Atlantic oligotrophic oceans. Because the shallower nitracline favored enhanced upward nitrate flux, the competitive advantage of cyanobacterial diazotrophs over nondiazotrophic phytoplankton was not as significant as it is in other oligotrophic oceans.

Analysis of the occurrence and activity of diazotrophic communities in organic and conventional horticultural soils

Nitrogen (N)-fixing microorganisms are an important soil component as they help replenish the pool of available N. Organic management can influence the N-fixing community; however, diazotroph community structure and activity in horticultural systems and the impacts of specific cultivation methods (i.e., greenhouse and open field) are unclear. Using the nifH gene, a marker gene for the microbial community involved in N fixation, we investigated the occurrence (DNA) and activity (RNA) of the diazotrophic community in organically and conventionally managed soils in a horticultural system over the course of 1 year. Ordination analysis of DGGE profiles revealed organic management affected the community structure in the greenhouse but not the open field; fertilization intensity may explain this divergent response, as indicated by the relevance of total C content to community structure. Quantitative PCR revealed that organic management increased the abundance and activity of diazotrophs. The soluble organic N concentration was higher in organically managed soils and during warmer months, and correlated with diazotroph abundance. Most identified sequences were from known diazotrophs, predominantly β-, γ- and α-proteobacteria. Twenty-four bands resembled Pseudomonas stutzeri and eight resembled Azoarcus sp. Our results show that the cultivation method controls the extent of the effects of season and organic management on diazotrophs, and that greenhouse cultivation can boost the effects of organic management on this community. Organic management intensified the positive effect of seasonal temperature on diazotroph abundance and activity, which may increase biological nitrogen fixation rates. In tandem, soil DNA and RNA analyses provide a comprehensive picture of the community.

Dynamics of soil diazotrophic community structure, diversity, and functioning during the cropping period of cotton (Gossypium hirsutum)

The soil sampled at different growth stages along the cropping period of cotton were analyzed using various molecular tools: restriction fragment length polymorphism (RFLP), terminal restriction length polymorphism (T-RFLP), and cloning-sequencing. The cluster analysis of the diazotrophic community structure of early sampled soil (0, 15, and 30 days) was found to be more closely related to each other than the later sampled one. Phylogenetic and diversity analysis of sequences obtained from the first (0 Day; C0) and last soil sample (180 day; C180) confirmed the data. The phylogenetic analysis revealed that C0 was having more unique sequences than C180 (presence of γ-Proteobacteria exclusively in C0). A relatively higher richness of diazotrophic community sequences was observed in C0 (S(ACE) : 30.76; S(Chao1) : 20.94) than C180 (S(ACE) : 18.00; S(Chao1) : 18.00) while the evenness component of Shannon diversity index increased from C0 (0.97) to C180 (1.15). The impact of routine agricultural activities was more evident based on diazotrophic activity (measured by acetylene reduction assay) than its structure and diversity. The nitrogenase activity of C0 (1264.85 ± 35.7 ηmol of ethylene production g(-1) dry soil h(-1) ) was statistically higher when compared to all other values (p < 0.05). There was no correlation found between diazotrophic community structure/diversity and N2 fixation rates. Thus, considerable functional redundancy of nifH was concluded to be existing at the experimental site.

Differing responses of marine N2 fixers to warming and consequences for future diazotroph community structure

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 72:33-46 (2014) - DOI: https://doi.org/10.3354/ame01683 Differing responses of marine N2 fixers to warming and consequences for future diazotroph community structure Fei-Xue Fu*, Elizabeth Yu, Nathan S. Garcia, Jasmine Gale, Yunsheng Luo, Eric A. Webb, David A. Hutchins Department of Biological Sciences, The University of Southern California, 3616 Trousdale Parkway, Los Angeles, California 90089, USA *Corresponding author: ffu@usc.edu ABSTRACT: The globally distributed colonial cyanobacterium Trichodesmium and unicellular diazotrophs including Crocosphaera together carry out the majority of marine biological nitrogen (N2) fixation. Future sea surface warming is predicted to influence their abundance and distribution, but temperature reaction norms have been determined for very few representatives of each genus. We compared thermal responses within and between the 2 genera Trichodesmium and Crocosphaera by measuring reaction norms for growth, N2 fixation, carbon fixation, and elemental ratios in 7 strains from a global culture collection. Temperature reaction norms of Trichodesmium and Crocosphaera were remarkably similar for all isolates within each genus, regardless of their geographic origin. Thermal limits of Trichodesmium and Crocosphaera ranged from 18 to 32°C and 24 to 32°C, and optimum growth temperatures were ~26 and ~30°C, respectively. The highest cellular ratios of nitrogen to phosphorus and carbon to nitrogen were found at optimum growth temperatures, and the lowest ratios near their thermal limits. In a mixed competition experiment, Trichodesmium growth rates were ~25% higher than those of Crocosphaera at 24°C, while those of Crocosphaera were ~50% higher at 28°C. Comparison of these results to current and projected seasonal temperature regimes in the subtropical Atlantic and Pacific Oceans suggests that predicted warmer temperatures may favor Crocosphaera over Trichodesmium, but that both genera may be excluded where future temperatures consistently exceed 32°C. Sea surface warming could profoundly alter the community structure and stoichiometry of marine N2-fixing cyanobacteria, thus fundamentally changing the biogeochemical cycling of this globally significant source of new nitrogen. KEY WORDS: Global change · Warming · Temperature · Nitrogen fixation · Trichodesmium · Crocosphaera Full text in pdf format PreviousNextCite this article as: Fu FX, Yu E, Garcia NS, Gale J, Luo Y, Webb EA, Hutchins DA (2014) Differing responses of marine N2 fixers to warming and consequences for future diazotroph community structure. Aquat Microb Ecol 72:33-46. https://doi.org/10.3354/ame01683 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 72, No. 1. Online publication date: March 07, 2014 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2014 Inter-Research.

Diazotroph community structure and abundance in wheat–fallow and wheat–pea crop rotations

Biological input of nitrogen (N) from the atmosphere by free-living diazotrophs can help alleviate fertilizer use in agricultural systems. In this study, we investigated the effect of N fertilizer and winter pea (Pisum sativum L.) crop on the community structure and abundance of free-living diazotrophs in a two year study of dryland winter wheat (Triticum aestivum L.) no-till production system in Eastern Oregon, USA. Based on quantification of the nifH gene, diazotroph abundance was strongly influenced by plant species and the crop year in which the soil samples were collected. A greater amount of nifH copies was recovered in 2012 compared to 2011 either as copies per gram soil or normalized to the abundance of bacterial 16S rRNA genes. The quantity of genes was greater under pea than wheat in 2012 although no difference was observed in the preceding year. The nifH gene abundance was positively correlated to ammonium concentration in 2011 and bacterial abundance in 2012. Nitrogen application did not influence diazotroph abundance in the top 0–5 cm; however the abundance was reduced by application at the lower 5–10 cm depth under wheat crop. The diazotroph community structure appeared to be influenced more by N fertilization rather than plant species with the exception of wheat in 2012. Changes in the community structure over the two years were greater for fertilized than unfertilized soil. Collectively, these data suggest that year-to-year variability had a greater influence on diazotroph communities rather than specific parameters of plant species, fertilization, total N, total organic C, or soil pH. Multi-year studies are necessary to define the specific drivers of diazotroph abundance, community structure and function.

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.

Response of biological soil crust diazotrophs to season, altered summer precipitation, and year-round increased temperature in an arid grassland of the colorado plateau, USA.

Biological soil crusts (biocrusts), which supply significant amounts of fixed nitrogen into terrestrial ecosystems worldwide (∼33 Tg y−1), are likely to respond to changes in temperature and precipitation associated with climate change. Using nifH gene-based surveys, we explored variation in the diazotrophic community of biocrusts of the Colorado Plateau, USA in response to season (autumn vs. spring), as well as field manipulations that increased the frequency of small volume precipitation events and year-round soil temperature. Abundance of nifH genes in biocrusts ranged from 3 × 106 to 1 × 108 g−1 soil, and nifH from heterocystous cyanobacteria closely related to Scytonema hyalinum, Spirirestis rafaelensis, and Nostoc commune comprised >98% of the total. Although there was no apparent seasonal effect on total nifH gene abundance in the biocrusts, T-RFLP analysis revealed a strong seasonal pattern in nifH composition. Spirirestis nifH abundance was estimated to oscillate 1 to >2 orders of magnitude between autumn (low) and spring (high). A year-round increase of soil temperature (2–3°C) had little effect on the diazotroph community structure over 2 years. Altered summer precipitation had little impact on diazotroph community structure over the first 1.5 years of the study, when natural background patterns across years and seasons superseded any treatment effects. However, after the second summer of treatments, nifH abundance was 2.6-fold lower in biocrusts receiving altered precipitation. Heterocystous cyanobacteria were apparently more resilient to altered precipitation than other cyanobacteria. The results demonstrate that diazotrophic community composition of biocrusts in this semi-arid grassland undergoes strong seasonal shifts and that the abundance of its dominant members decreased in response to more frequent, small volume precipitation events.

Diversity of nifH gene sequences in the sediments of South China Sea

In order to contribute to knowledge about structure of marine diazotrophic communities in the sediments of South China Sea, the molecular diversity of the nifH gene, which encodes the Fe protein of the nitrogenase complex, was assessed by polymerase chain reaction (PCR) amplification using PolF/R primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental deoxyribonucleic acid (DNA) samples collected during three stations including shallow sea (75 m, station L10), shelf (450 m, station L2) and deep sea (1000 m, station L21), and covering an area between 17 to 19°N and 111 to 119°E. Samples from shallow sea contained β-, and δ- proteobacteria; the shelf contained α-, β-, δ- proteobacteria; the deep sea contained α-, δ-, γ-proteobacteria, firmicutes, and green nonsulfur (GNS) bacterium. These results suggested that diazotroph was significant component potentially contributing to nitrogen fixation in South China Sea. Key words: nifH, diversity, sediment, the South China Sea.

Long‐term nitrogen and phosphorus fertilization effects on N2 fixation rates and nifH gene community patterns in mangrove sediments

AbstractThe bioavailability of nutrients is important in controlling ecological processes and nitrogen cycling in oligotrophic mangrove forests, yet the variation of diazotrophic community structure and activity with nutrient availability in sediments remains largely unexplored. To investigate for the first time how nutrients in sediments affect spatial and temporal patterns of diazotrophic community structure and activity, the sedimentary environment of Twin Cays, Belize, was examined with respect to the effects of long‐term fertilization [treatments: control (Ctrl), nitrogen (N), and phosphorus (P)] on N2 fixation rates and nifH gene community structure. We found that N2 fixation rates were significantly higher at the P‐treatment, intermediate at the Ctrl‐treatment and lower in the N‐treatment (P: 4.2 ± 0.5, Crtl: 0.8 ± 0.1, N: 0.4 ± 0.1 nmol·N·g−1·h−1; P &lt; 0.001) with spatial (Ctrl‐ and P‐treatments) and temporal (only P‐treatment) variability positively correlated with live root abundance (r2 = 0.473, P &lt; 0.001) and concentration (r2 = 0.458, P &lt; 0.0001). The community structure of diazotrophs showed larger spatial and temporal variability in the fertilized treatments than in the Ctrl‐treatment, with the relative abundance of OTUs (nifH operational taxonomic units) at the fertilized treatments inversely related to live root abundance. Overall, long‐term fertilization (with either N or P) affects not only nutrient levels in mangrove sediments directly, but also spatial and temporal patterns of both community structure and activity and likely plant‐microbe interactions as well. Our findings suggest that the maintenance of natural nutrient conditions in mangrove sediments is important to ensure the stability of microbial functional groups like diazotrophs.

Regional and local factors affecting diversity, abundance and activity of free-living, N2-fixing bacteria in Australian agricultural soils

N2-fixation by free-living (diazotrophic) microorganism is a key process affecting ecosystem functioning in soils. Understanding drivers affecting diazotrophic community assemblages and activities may lead to management practices to increase primary production and/or environmental sustainability. We used PCR-DGGE to determine the fundamental relationships between diazotrophic community structure and in a wide range of soils across southern Australia. In addition qPCR, RT-qPCR and N2-fixation (acetylene reduction) were used to investigate factors influencing gene abundance, expression and processes in similar soils with different agricultural inputs. Across 22 soils, the structural composition of the nifH community was significantly influenced by site (ANOSIM R = 0.876; P = 0.001). The effects of management practices were evident, and often larger than between-soil differences, but were only present at some sites. Differences in nifH communities between sites correlated to particulate organic carbon (POC; measured by mid-infrared spectroscopy) content of the soils (BIO-ENV test; ρ = 0.502; P = 0.001), but not other factors including total soil C. In 3 soils from the Murrumbidgee irrigation region of NSW, intensification of the farming systems was associated with increasing N2-fixation (P < 0.05), except where rice was cultivated. N2-fixation correlated either with nifH abundance or gene expression in soils, but not both. Our data shows that soil C is closely linked to diazotrophic ecology. Principally, the amount of C entering the soil system is directly related to the abundance and N2-fixation activity of free-living bacteria. However, we also show that C in the POC pool has associative links to the genetic diversity of the soil diazotroph community. Given the importance of diversity and abundance of functional organisms in supporting ecosystem processes, we suggest that soil C inputs should be considered for both qualitative and quantitative properties when considering impacts on diazotrophic bacterial ecology.

Physical forcing of nitrogen fixation and diazotroph community structure in the North Pacific subtropical gyre

Dinitrogen (N2) fixing microorganisms (termed diazotrophs) exert important control on the ocean carbon cycle. However, despite increased awareness on the roles of these microorganisms in ocean biogeochemistry and ecology, the processes controlling variability in diazotroph distributions, abundances, and activities remain largely unknown. In this study, we examine 3 years (2004–2007) of approximately monthly measurements of upper ocean diazotroph community structure and rates of N2 fixation at Station ALOHA (22°45′N, 158°W), the field site for the Hawaii Ocean Time‐series program in the central North Pacific subtropical gyre (NPSG). The structure of the N2‐fixing microorganism assemblage varied widely in time with unicellular N2‐fixing microorganisms frequently dominating diazotroph abundances in the late winter and early spring, while filamentous microorganisms (specifically various heterocyst‐forming cyanobacteria and Trichodesmium spp.) fluctuated episodically during the summer. On average, a large fraction (∼80%) of the daily N2 fixation was partitioned into the biomass of &lt;10 μm microorganisms. Rates of N2 fixation were variable in time, with peak N2 fixation frequently coinciding with periods when heterocystous N2‐fixing cyanobacteria were abundant. During the summer months when sea surface temperatures exceeded 25.2°C and concentrations of nitrate plus nitrite were at their annual minimum, rates of N2 fixation often increased during periods of positive sea surface height anomalies, as reflected in satellite altimetry. Our results suggest mesoscale physical forcing may comprise an important control on variability in N2 fixation and diazotroph community structure in the NPSG.

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

The diazotrophic communities in a rice paddy field were characterized by a molecular polyphasic approach including DNA/RNA-DGGE fingerprinting, real time RT-PCR analysis of nifH gene and the measurement of nitrogen fixation activities. The investigation was performed on a diurnal cycle and comparisons were made between bulk and rhizosphere / root soil as well as between fertilized / unfertilized soils. Real time RT-PCR showed no significant difference in the total quantity of nifH expression under the conditions investigated. The functional diversity and dynamics of the nifH gene expressing diazotroph community investigated using RT-PCR-DGGE revealed high diurnal variations, as well as variation between different soil types. Most of the sequence types recovered from the DGGE gels and clone libraries clustered within nifH Cluster I and III (65 different nifH sequences in total). Sequence types most similar to Azoarcus spp., Metylococcus spp., Rhizobium spp., Methylocystis spp., Desulfovibrio spp., Geobacter spp., Chlorobium spp., were abundant and indicate that these species may be responsible for the observed diurnal variation in the diazotrophic community structure in these rice field samples. Previously described diazotrophic cyanobacterial genera in rice fields, such as Nostoc and Cyanothece, were present in the samples but not detectable in RT-PCR assays.

The communities of endophytic diazotrophic bacteria in cultivated rice ( Oryza sativa L .)

Even though attempts have been made to use endophytic diazotrophic bacteria as biofertilizers for rice cultivation, their community structure and complexity in rice tissues is not well understood. In this study, the diversity and the community structure of endophytic diazotrophs as well as the nifH gene expression within each plant part and growth stage of rice under different soil conditions were investigated. The population of viable endophytic diazotrophic bacteria in cultivated rice ( Oryza sativa L. cultivar KDML-105) was investigated under different soil conditions and the N 2-fixing ability of selected consortia and single isolates was assessed. Single isolates from each diazotrophic consortium were shown to be capable of both the inhibition and promotion of N 2-fixation. Some isolates were closely related to Enterobacter dissolvens, Brevundimonas aurantiaca, Pantoea agglomerans, Pseudomonas spp., and Enterobacteriaceae. Carbon source utilization, and production of IAA, pectinase and cellulase indicated high diversity in the rice tissues. The presence of diazotrophic bacteria was detected in roots, stems and leaves on the basis of the GUS reporter gene. PCR-DGGE analysis conducted directly on rice tissue samples using 16S rDNA primers was used to elucidate the structure of the endophytic bacterial communities. Nested PCR-DGGE analysis with nifH primer demonstrated less diazotrophic bacterial diversity in the roots of rice cultivated in paddy soil amended with nitrogen fertilizer than in unfertilized and previously uncultivated soil. Plant tissue type was found to dictate the endophytic diazotrophic community structure rather than the type of soil or fertilizer amendment. In order to detect the nifH gene expression which implies the nitrogen fixing activities of the diazotrophic bacterial community, the RT-PCR approach was used. The results demonstrated that nifH gene expression could be differently detected in each part and growth stage of rice plants as well as being influenced by soil nitrogen status. The expression level of the nifH gene in all roots from plants grown in N-fertilized soil was the lowest among the treatments studied. The results confirm the complexity of the endophytic diazotrophic bacterial community, and indicate that the type of plant tissue seems to influence the community structure.

Analysis of nifH gene diversity in red soil amended with manure in Jiangxi, south China

In order to understand the community structure of diazotrophs in red soil and effects of organic manure Application on the structure, four nifH gene libraries were constructed: the control (CK), low manure (LM), High manure (HM), and high manure adding lime (ML). Totally 150 nifH gene clones were screened and grouped into 21 clusters by RFLP analysis. Existence of dominant patterns was observed in all libraries, which counted for over 96% of clones in library HM and about 56 approximately 72% in other three libraries. The nifH sequences of the dominant patterns in all libraries were most similar to sequences of the cyanobacteria. nifH genes showed high diversity in red soil, dispersing throughout the nifH clades (alpha-, beta-, and gamma-Proteobacteria, Firmicutes, cyanobacteria, Verrucomicrobia, and posited group). Bradyrhizobium and Burkholderia were also important diaxotrophs in low fertility soil samples. Low manure treatment increased the Diversity of nifH genes compared with CK and high manure treatments. Manure and lime treatment led to obvious community succession. Total N to available P ratio, total carbon, and K concentrations were the main factors affecting the diversity of diazotrophs in red soil.