Diazotrophic Community Composition Research Articles

Fronts divide diazotroph communities in the Southern Indian Ocean.

Dinitrogen (N2) fixation represents a key source of reactive nitrogen in marine ecosystems. While the process has been rather well explored in low latitudes of the Atlantic and Pacific Oceans, other higher latitude regions and particularly the Indian Ocean have been chronically overlooked. Here we characterize N2 fixation and diazotroph community composition across nutrient and trace metals gradients spanning the multi-frontal system separating the oligotrophic waters of the Indian Ocean subtropical gyre from the high nutrient low chlorophyll waters of the Southern Ocean. We found a sharp contrasting distribution of diazotroph groups across the frontal system. Notably, cyanobacterial diazotrophs dominated north of fronts, driving elevated N2 fixation rates (up to 13.96nmol N l-1 d-1) with notable peaks near the South African coast. South of the fronts non-cyanobacterial diazotrophs prevailed without significant N2 fixation activity being detected. Our results provide new crucial insights into high latitude diazotrophy in the Indian Ocean, which should contribute to improved climate model parameterization and enhanced constraints on global net primary productivity projections.

Nitrogen-fixing bacterial communities differ between perennial agroecosystem crops.

Biocrusts, common in natural ecosystems, are specific assemblages of microorganisms at or on the soil surface with associated microorganisms extending into the top centimeter of soil. Agroecosystem biocrusts have similar rates of nitrogen (N) fixation as those in natural ecosystems, but it is unclear how agricultural management influences their composition and function. This study examined the total bacterial and diazotrophic communities of biocrusts in a citrus orchard and a vineyard that shared a similar climate and soil type but differed in management. To contrast climate and soil type, these biocrusts were also compared with those from an apple orchard. Unlike natural ecosystem biocrusts, these agroecosystem biocrusts were dominated by proteobacteria and had a lower abundance of cyanobacteria. All of the examined agroecosystem biocrust diazotroph communities were dominated by N-fixing cyanobacteria from the Nostocales order, similar to natural ecosystem cyanobacterial biocrusts. Lower irrigation and fertilizer in the vineyard compared with the citrus orchard could have contributed to biocrust microbial composition, whereas soil type and climate could have differentiated the apple orchard biocrust. Season did not influence the bacterial and diazotrophic community composition of any of these agroecosystem biocrusts. Overall, agricultural management and climatic and edaphic factors potentially influenced the community composition and function of these biocrusts.

Organic amendments increased Chinese milk vetch symbiotic nitrogen fixation by enriching Mesorhizobium in rhizosphere.

Symbiotic nitrogen fixation of Chinese milk vetch (Astragalus sinicus L.) can fix nitrogen from the atmosphere and serve as an organic nitrogen source in agricultural ecosystems. Exogenous organic material application is a common practice of affecting symbiotic nitrogen fixation; however, the results of the regulation activities remain under discussion. Studies on the impact of organic amendments on symbiotic nitrogen fixation have focused on dissolved organic carbon content changes, whereas the impact on dissolved organic carbon composition and the underlying mechanism remain unclear. In situ pot experiments were carried out using soils from a 40-year-old field experiment platform to investigate symbiotic nitrogen fixation rate trends, dissolved organic carbon concentration and component, and diazotroph community structure in roots and in rhizosphere soils following long-term application of different exogenous organic substrates, i.e., green manure, green manure and pig manure, and green manure and rice straw. Remarkable increases in rate were observed in and when compared with that in green manure treatment, with the greatest enhancement observed in the treatment. Moreover, organic amendments, particularly pig manure application, altered diazotroph community composition in rhizosphere soils, therefore increasing the abundance of the host-specific genus Mesorhizobium. Furthermore, organic amendments influence the diazotroph communities through two primary mechanisms. Firstly, the components of dissolved organic carbon promote an increase in available iron, facilitated by the presence of humus substrates. Secondly, the elevated content of dissolved organic carbon and available iron expands the niche breadth of Mesorhizobium within the rhizosphere. Consequently, these alterations result in a modified diazotroph community within the rhizosphere, which in turn influences Mesorhizobium nodulation in the root and symbiotic nitrogen fixation rate. The results of the present study enhance our understanding of the impact of organic amendments on symbiotic nitrogen fixation and the underlying mechanism, highlighting the key role of dissolved organic carbon composition on diazotroph community composition in the rhizosphere.

Diazotrophic community in the sediments of Poyang Lake in response to water level fluctuations.

Water level fluctuations (WLFs) are typical characteristic of floodplain lakes and dominant forces regulating the structure and function of lacustrine ecosystems. The sediment diazotrophs play important roles in contributing bioavailable nitrogen to the aquatic environment. However, the relationship between the diazotrophic community and WLFs in floodplain lakes is unknown. In this paper, we carried out a comprehensive investigation on the alpha diversity, abundance, composition and co-occurrence network of the sediment diazotrophs during different water level phases in Poyang Lake. There were no regular variation patterns in the alpha diversity and abundance of the sediment diazotrophs with the water level phase transitions. The relative abundance of some diazotrophic phyla (including Alphaproteobacteria, Deltaproteobacteri, Euryarchaeota, and Firmicutes) and genera (including Geobacter, Deferrisoma, Desulfuromonas, Rivicola, Paraburkholderia, Methylophilus, Methanothrix, Methanobacterium, and Clostridium) was found to change with the water level phase transitions. The results of ANOSIM, PerMANOVA, and DCA at the OTU level showed that the diazotrophic community structure in the low water level phase was significantly different from that in the two high water level phases, while there was no significant difference between the two high water level phases. These results indicated that the diazotrophic community was affected by the declining water level in terms of the composition, while the rising water level contributed to the recoveries of the diazotrophic community. The diazotrophs co-occurrence network was disrupted by the declining water level, but it was strengthened by the rising water level. Moreover, redundancy analysis showed that the variation of the diazotrophic community composition was mostly related to sediment total nitrogen (TN) and total phosphorous (TP). Interestingly, the levels of sediment TN and TP were also found to vary with the water level phase transitions. Therefore, it might be speculated that the WLFs may influence the sediment TN and TP, and in turn influence the diazotrophic community composition. These data can contribute to broadening our understanding of the ecological impacts of WLFs and the nitrogen fixation process in floodplain lakes.

Phosphorus availability regulates nitrogen fixation rate through a key diazotrophic assembly: Evidence from a subtropical Moso bamboo forest subjected to nitrogen application

Biological N fixation (BNF) is an important N input process for terrestrial ecosystems. Long-term N application increases the availability of N, but may also lead to phosphorus (P) deficiency or an imbalance between N and P. Here, we performed a 5-year N application experiment in a subtropical Phyllostachys heterocycla forest in site and a P application experiment in vitro to investigate the effect of N application on the BNF rate and its regulatory factor. The BNF rate, nifH gene, free-living diazotrophic community composition and plant properties were measured. We found that N application suppressed the BNF rate and nifH gene abundance, whereas the BNF rate in soils with added P was significantly higher overall than that in soils without added P. Moreover, we identified a key diazotrophic assembly (Mod#2), primarily comprising Bradyrhizobium, Geobacter, Desulfovibrio, Anaeromyxobacter, and Pseudodesulfovibrio, which explained 77 % of the BNF rate variation. There was a significant positive correlation between the Mod#2 abundance and soil available P, and the random forest results showed that soil available P is the most important factor affecting the Mod#2 abundance. Our findings highlight the importance of soil P availability in regulating the activities of key diazotrophs, and thus increasing P supply may help to promote N accumulation and primary productivity through facilitating the BNF process in forest ecosystems.

Tree species-specific wood traits control diazotrophic community composition in deadwood

Deadwood is a nitrogen (N) poor environment and its decomposition rate is limited by low N bioavailability. Diazotrophs reduce such N limitations as they are capable of fixing N2 which employs the functional gene nifH. The present study investigates the diazotrophic community composition in deadwood logs of 13 different tree species in temperate forests. Directly after sampling, deadwood was incubated with water (total fraction) or bromodeoxyuridine (BrdU), and latter labeled the metabolically active fraction. Nucleic acid extracts from both fractions were used to assess the nifH gene-based diversity by amplicon sequencing. The active fraction was a subset of the total fraction, which differed in abundance and with a lower richness. Wood traits such as phosphorous, molybdenum, magnesium, glucose concentration, and C:N ratio were positive factors of richness and community composition of the active fraction. Bradyrhizobium and Methylocapsa dominated the total fraction, while the active fraction was mainly governed by Frankia and Treponema regardless of tree species studied, suggesting that they are well adapted to C sources and nutrient-poor conditions in deadwood. The great diazotrophic diversity (50 genera) in the active fraction indicates an enormous potential for biological N fixation during deadwood decay.

Rhizoid-associated cyanobacteria are the main contributors to biological nitrogen fixation in restored moss crust

Biological soil crusts (biocrusts) are associations of highly specialized communities and topsoil particles and play important ecosystem functions in drylands. Biotic interactions affected the assemblage of biocrust communities and thus their ecological function and adaption abilities. As the dominant organism in the moss crust, moss plays a significant role in modifying the microenvironment and releasing carbon, and this may affect the distribution and metabolic activity of diazotrophic cyanobacteria, which are the dominant diazotrophs. In this study, we examined the contribution of diazotrophic cyanobacteria to nitrogenase activity (NA) by investigating the NA under different light conditions and estimated the proportion of diazotrophic cyanobacteria from total diazotrophs. We tested the spatial distribution of cyanobacteria using laser confocal fluorescence microscopy (CLSM). Additionally, we separated diazotrophs in moss crust into free-living and moss-associated diazotrophs and compared the NA, nifH gene abundances and their transcripts, and the community composition of free-living and moss-associated diazotrophs. The light-dependent characteristic of NA and dominance of heterocystous cyanobacteria (accounting for over 90% of total nifH sequences at both the DNA and transcript levels) suggested a significant contribution of heterocystous cyanobacteria to NA. Cyanobacteria were mainly distributed in a few millimeters of subsoil in which abundant cyanobacteria were associated with moss rhizoids. The NA rates of moss-associated diazotrophs were comparable to those of moss crust and approximately 4–8 times higher than those of free-living diazotrophs, indicating the major role of moss-associated diazotrophs in nitrogen fixation. The community composition of moss-associated diazotrophs was similar to that of free-living diazotrophs. Moss-associated diazotrophs have a higher NA per copy of the nifH gene and transcript than free-living diazotrophs, proving the synergistic association of moss and cyanobacteria with NA.

Soil aggregate size mediates the impact of different fertilization patterns on the diazotrophic community of mine soils

The development of the mining industry has led to significant environmental pollution around mining sites, affecting the nitrogen (N) cycle and associated microorganisms and disrupting the structure of soil aggregates. In this study, mine soil incubation experiments were conducted under the application of two types of N fertilizer (urea (U) and ammonium chloride (AC)) and nine different fertilization patterns, and the effects of the different N fertilization patterns on soil aggregate properties and diazotrophic community structure were compared. The results showed that carbon (C), phosphorus (P) and N were mainly enriched in megaaggregates (2–4 mm) and macroaggregates (0.25–2 mm). N fertilizer addition decreased the bioavailable heavy metal (HM) content of soil aggregates to different degrees. In addition, N fertilization resulted in significantly higher activities of cellulase, invertase, amylase, urease, protease and phosphatase in microaggregates (<0.25 mm) than in mega- and macroaggregates (p < 0.05). Among the nine fertilization patterns, AC application was performed at a low frequency and high concentration. In most cases, U application resulted in a higher nifH gene abundance than AC application. Specifically, nifH gene abundance in microaggregates in the four U treatments ranged from 9.75 × 106 to 16.63 × 106 gene copies g−1 dry soil, which was significantly higher than that in the control (p < 0.05). Proteobacteria was the major diazotrophic phylum, accounting for 52.9 % to 79.1 %, 46.4 % to 80.3 % and 49.1 % to 81.2 % in mega-, macro- and microaggregates, respectively, followed by Firmicutes and Actinobacteria. The application of N fertilizers had different impacts on soil physicochemical properties, which in turn indirectly affected the abundance and community composition of soil diazotrophs. Among the aggregate properties, microbial nitrogen (MBN) was an important variable that explained the variation in diazotrophic communities (p < 0.05). Our results suggest that the addition of N fertilizer affects the physicochemical properties and the structure and functions of diazotrophic community in soil aggregates.

Nitrogen fixation and diazotroph diversity in groundwater systems.

Biological nitrogen fixation (BNF), the conversion of N2 into bioavailable nitrogen (N), is the main process for replenishing N loss in the biosphere. However, BNF in groundwater systems remains poorly understood. In this study, we examined the activity, abundance, and community composition of diazotrophs in groundwater in the Hetao Plain of Inner Mongolia using 15N tracing methods, reverse transcription qPCR (RT-qPCR), and metagenomic/metatranscriptomic analyses. 15N2 tracing incubation of near in situ groundwater (9.5-585.4 nmol N L-1 h-1) and N2-fixer enrichment and isolates (13.2-1728.4 nmol N g-1 h-1, as directly verified by single-cell resonance Raman spectroscopy), suggested that BNF is a non-negligible source of N in groundwater in this region. The expression of nifH genes ranged from 3.4 × 103 to 1.2 × 106 copies L-1 and was tightly correlated with dissolved oxygen (DO), Fe(II), and NH4+. Diazotrophs in groundwater were chiefly aerobes or facultative anaerobes, dominated by Stutzerimonas, Pseudomonas, Paraburkholderia, Klebsiella, Rhodopseudomonas, Azoarcus, and additional uncultured populations. Active diazotrophs, which prefer reducing conditions, were more metabolically diverse and potentially associated with nitrification, sulfur/arsenic mobilization, Fe(II) transport, and CH4 oxidation. Our results highlight the importance of diazotrophs in subsurface geochemical cycles.

Stochastic Processes Dominate in the Water Mass-Based Segregation of Diazotrophs in a High Arctic Fjord (Svalbard).

Nitrogen-fixing or diazotrophic microbes fix atmospheric nitrogen (N2) to ammonia (NH3+) using nitrogenase enzyme and play a crucial role in regulating marine primary productivity and carbon dioxide sequestration. However, there is a lack of information about the diversity, structure, and environmental regulations of the diazotrophic communities in the high Arctic fjords, such as Kongsfjorden. Here, we employed nifH gene sequencing to clarify variations in composition, community structure, and assembly mechanism among the diazotrophs of the salinity-driven stratified waters of Kongsfjorden. The principal environmental and ecological drivers of the observed variations were identified. The majority of the nifH gene sequences obtained in the present study belonged to cluster I and cluster III nifH phylotypes, accounting for 65% and 25% of the total nifH gene sequences. The nifH gene diversity and composition, irrespective of the size fractions (free-living and particle attached), showed a clear separation among water mass types, i.e., Atlantic-influenced versus glacier-influenced water mass. Higher nifH gene diversity and relative abundances of non-cyanobacterial nifH OTUs, affiliated with uncultured Rhizobiales, Burkholderiales, Alteromonadaceae, Gallionellaceae (cluster I) and uncultured Deltaproteobacteria including Desulfuromonadaceae (cluster III), were prevalent in GIW while uncultured Gammaproteobacteria and Desulfobulbaceae were abundant in AIW. The diazotrophic community assembly was dominated by stochastic processes, principally ecological drift, and to lesser degrees dispersal limitation and homogeneous dispersal. Differences in the salinity and dissolved oxygen content lead to the vertical segregation of diazotrophs among water mass types. These findings suggest that water column stratification affects the composition and assembly mechanism of diazotrophic communities and thus could affect nitrogen fixation in the Arctic fjord.

Biochar amendment increases the abundance and alters the community composition of diazotrophs in a double rice cropping system

Biochar amendment increases the abundance and alters the community composition of diazotrophs in a double rice cropping system

Glacial meltwater and seasonality influence community composition of diazotrophs in Arctic coastal and open waters.

The Arctic Ocean is particularly affected by climate change with unknown consequences for primary productivity. Diazotrophs-prokaryotes capable of converting atmospheric nitrogen to ammonia-have been detected in the often nitrogen-limited Arctic Ocean but distribution and community composition dynamics are largely unknown. We performed amplicon sequencing of the diazotroph marker gene nifH from glacial rivers, coastal, and open ocean regions and identified regionally distinct Arctic communities. Proteobacterial diazotrophs dominated all seasons, epi- to mesopelagic depths and rivers to open waters and, surprisingly, Cyanobacteria were only sporadically identified in coastal and freshwaters. The upstream environment of glacial rivers influenced diazotroph diversity, and in marine samples putative anaerobic sulphate-reducers showed seasonal succession with highest prevalence in summer to polar night. Betaproteobacteria (Burkholderiales, Nitrosomonadales, and Rhodocyclales) were typically found in rivers and freshwater-influenced waters, and Delta- (Desulfuromonadales, Desulfobacterales, and Desulfovibrionales) and Gammaproteobacteria in marine waters. The identified community composition dynamics, likely driven by runoff, inorganic nutrients, particulate organic carbon, and seasonality, imply diazotrophy a phenotype of ecological relevance with expected responsiveness to ongoing climate change. Our study largely expands baseline knowledge of Arctic diazotrophs-a prerequisite to understand underpinning of nitrogen fixation-and supports nitrogen fixation as a contributor of new nitrogen in the rapidly changing Arctic Ocean.

Soil phosphorus drives variation in diazotrophic communities in a subtropical nitrogen-rich forest

Although nitrogen (N)-fixing bacteria (diazotrophs) play a vital role in the biosphere's N cycle. However, large uncertainty exists regarding how atmospheric N deposition regulates biological N fixation (BNF) in soils and which diazotrophic taxa are responsible for this process. We targeted the nifH gene to investigate the effects of long-term N addition on the abundance and composition of diazotrophic communities in subtropical N-rich forests, using real-time quantitative polymerase chain reaction (q-PCR) and MiSeq sequencing. We conducted a field trial for seven years and included three N treatment levels (+0, +50, +150 kg N ha−1 yr−1; CK, LN, HN). With increasing N addition, the average total P concentration in the whole soil profile and the topsoil P availability decreased significantly, further reducing nitrogenase activity and nifH abundance significantly, while increasing the diversity of the diazotrophic community. After seven years of N addition, the relative abundance of uncultured Cyanobacteria significantly decreased, while those of Rhizobiales and Desulfobulbaceae significantly increased. The diazotrophic communities transitioned from free-living N fixers (uncultured Cyanobacteria-dominated) to symbiotic N fixers (Rhizobiales-dominated; HN treatment). The relative abundance of heterotrophic diazotrophic bacteria and the occasional diazotrophic species increased, resulting in diazotrophic bacteria still maintaining high competitiveness in the N-rich and/or P-limited soils. Proteobacteria (e.g., Rhizobiales) had the highest potential to participate in microbial mechanisms triggered by P scarcity. Variation partitioning analyses showed that diazotrophic community composition and diversity were mainly correlated with the soil P availability, with soil AP having the highest individual effect. Overall, this study will broaden our understanding of N fixation mechanisms and the diazotrophic community variation in N-rich forests, while also highlighting soil P as a key factor driving changes in diazotrophic communities.

Seasonal and spatial patterns in diazotroph community composition at Station ALOHA

Dinitrogen (N2) fixation is carried out by specialized microbes, called diazotrophs, and is a major source of nitrogen supporting primary production in oligotrophic oceans. One of the best-characterized diazotroph habitats is the North Pacific Subtropical Gyre (NPSG), where warm, chronically N-limited surface waters promote year-round N2 fixation. At Station ALOHA (A Long-Term Oligotrophic Habitat Assessment) in the NPSG, N2 fixation is typically ascribed to conspicuous, filamentous cyanobacterial diazotrophs (Trichodesmium and Richelia), unicellular free-living Crocosphaera, and the UCYN-A/haptophyte symbiosis, based on using microscopy and quantitative PCR (qPCR). However, the diazotroph community in this ecosystem is diverse and includes non-cyanobacterial diazotrophs (NCDs). We investigated the diversity, depth distributions, and seasonality of diazotroph communities at Stn. ALOHA using high throughput sequencing (HTS) of nifH gene fragments from samples collected throughout the euphotic zone (0-175 m) at near-monthly intervals from June 2013 to July 2016. The UCYN-A symbioses and Trichodesmium sp. consistently had the highest relative abundances and seasonal patterns that corroborated qPCR-based analyses. Other prevalent community members included a new Crocosphaera-like species, and several NCDs affiliated with γ- and δ-proteobacteria. Notably, some of the NCDs appear to be stable components of the community at Stn. ALOHA, having also been reported in prior studies. Depth and temporal patterns in microdiversity within two major diazotroph groups (Trichodesmium and UCYN-A) suggested that sub-populations are adapted to time- and depth-dependent environmental variation. A network analysis of the upper euphotic (0-75 m) HTS data identified two modules that reflect a diazotroph community structure with seasonal turnover between UCYN-A/Gamma A, and Trichodesmium/Crocosphaera. It also reveals the seasonality of several important cyanobacteria and NCDs about which little is known, including a putative δ-proteobacterial phylotype originally discovered at Stn. ALOHA. Collectively, these results underscore the importance of coupling nifH gene HTS with other molecular techniques to obtain a comprehensive view of diazotroph community composition in the marine environment and reveal several understudied diazotroph groups that may contribute to N2 fixation in the NPSG.

Control factors of soil diazotrophic community assembly and nitrogen fixation rate across eastern China

Biological nitrogen (N) fixation is an essential N input into terrestrial ecosystems and plays a critical role in global N cycling. However, the driving factors of the diazotrophic community composition and soil N fixation rate at the regional scale remain largely unclear, especially in different land-use types. Here, we explored the environmental adaptation of diazotrophic communities and controlling factors of N fixation in maize, rice, and forest soils across eastern China. Results showed that the diversity, composition, and co-occurrence network differed significantly among these habitats, with a relatively higher alpha diversity and network complexity in rice fields. The mean annual precipitation plays a predominant role in shaping diazotrophic community composition. Dispersal limitation, belonging to stochastic processes, contributed most to diazotrophic community assembly in terrestrial ecosystems. Habitat niche breadth analysis indicated that the environmental adaptation of diazotrophic communities was highest in rice fields, followed by forest and maize fields. The overall N fixation rate was higher in rice fields, followed by maize and forest soils. Structural equation modeling revealed that the N fixation rate was mainly affected by soil properties in maize and forest soils. In contrast, the N fixation rate was more influenced by diazotrophic community composition and climatic factors in rice habitats. Our results revealed that the environmental adaptation of the diazotrophic communities and controlling factors of the N fixation function were different among maize, rice, and forest ecosystems. These findings will help us understand and predict the N bioavailability in different ecosystem types under climate changes.

Alterations in substrate stoichiometry control the responses of soil diazotrophs to nutrient enrichment

Soil diazotrophs convert atmospheric nitrogen (N) into biologically active N, which is an important N source for plants and microbes in many natural ecosystems. However, responses of diazotrophic communities to long-term N and phosphorus (P) inputs and the primary mediators remain unclear, particularly in semi-arid grasslands. Here we show that the abundance and community composition of diazotrophs responded distinctly to gradients of long-term N and P additions in an Inner Mongolia steppe. Our results showed that moderate to high N inputs (105–280 kg N ha−1 yr−1) significantly reduced both diazotrophic abundance and community diversity. In contrast, low to moderate P additions (20–80 kg P2O5 ha−1 yr−1), along with low N input (24 kg N ha−1 yr−1), significantly increased diazotrophic abundance, while having no significant effects on soil diazotrophic diversity and community structure. Also, long-term N and P inputs led to different co-occurrence patterns in the diazotrophic community, although both of them reduced the stability of diazotrophic co-occurrence network. Structural equation modeling (SEM) analysis further revealed that N additions suppressed the diazotrophic abundance and diversity, corresponding to the decreasing stoichiometric ratio of soil available C to N. Together, these results indicate that nutrient-induced changes in the relative availability of C to N in soil primarily drives the impacts of nutrient enrichment on diazotrophs. These findings suggest that management regimes with low nutrient inputs may provide unique opportunities to increase plant productivity, while sustaining or even enhancing the abundance and activity of soil diazotrophs.

The diversity and structure of diazotrophic communities in the rhizosphere of coastal saline plants is mainly affected by soil physicochemical factors but not host plant species

The diversity and community structure of rhizospheric microbes are largely affected by soil physicochemical properties and plant species. In this work, high throughput sequencing and quantitative real-time PCR targeting nifH gene were used to assess the abundance and diversity of diazotrophic community in the coastal saline soils of Yellow River Delta (YRD). We demonstrated that the copy number of nifH gene encoding the Fe protein subunit of the nitrogenase in the nitrogen fixation process was significantly affected by soil physiochemical factors, and the abundance of diazotrophs in the rhizospheric soil samples collected from different locations was positively related with soil physicochemical properties. Soil salinity (P=0.003) and moisture (P=0.003) were significantly co-varied with the OTU-based community composition of diazotrophs. Taxonomic analysis showed that most diazotrophs belonged to the Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. Linear discriminant analysis (LDA) effect size (LEfSe) and canonical correspondence analysis (CCA) showed that diazotrophic community structure significantly varied with soil salinity, moisture, pH and total nitrogen, carbon, sulphur and nitrite (NO2–N) content. Our findings provide direct evidence toward the understanding of different effects of soil physicochemical properties and host plant traits such as halophytes types, life span and cotyledon type, on the community composition of diazotrophic populations in the rhizosphere of plants grown in coastal saline soils.

Temporal dynamics of total and active root-associated diazotrophic communities in field-grown rice

Plant-associated nitrogen-fixing microorganisms (diazotrophs) are essential to host nutrient acquisition, productivity and health, but how host growth affects the succession characteristics of crop diazotrophic communities is still poorly understood. Here, Illumina sequencing of DNA- and RNA-derived nifH genes was employed to investigate the dynamics of total and active diazotrophic communities across rhizosphere soil and rice roots under four fertilization regimes during three growth periods (tillering, heading and mature stages) of rice in 2015 and 2016. Our results indicated that 71.9–77.2% of the operational taxonomic units (OTUs) were both detected at the DNA and RNA levels. According to the nonmetric multidimensional scaling ordinations of Bray–Curtis distances, the variations in community composition of active rhizosphere diazotrophs were greater than those of total rhizosphere diazotrophs. The community composition (β-diversity) of total and active root-associated diazotrophs was shaped predominantly by microhabitat (niche; R2 ≥ 0.959, p < 0.001), followed by growth period (R2 ≥ 0.15, p < 0.001). The growth period had a stronger effect on endophytic diazotrophs than on rhizosphere diazotrophs. From the tillering stage to the heading stage, the α-diversity indices (Chao1, Shannon and phylogenetic diversity) and network topological parameters (edge numbers, average clustering coefficient and average degree values) of total endophytic diazotrophic communities increased. The proportions of OTUs shared by the total rhizosphere and endophytic diazotrophs in rhizosphere diazotrophs gradually increased during rice growth. Moreover, total diazotrophic α-diversity and network complexity decreased from rhizosphere soil to roots. Collectively, compared with total diazotrophic communities, active diazotrophic communities were better indicators of biological response to environmental changes. The host microhabitat profoundly drove the temporal dynamics of total and active root-associated diazotrophic communities, followed by the plant growth period.

Changes in community assembly processes and co-occurrence networks of soil diazotrophs along an elevational gradient in Tibetan alpine meadows

Shifts in soil microbial community composition along elevational gradients have been extensively studied; yet, the assembly process and co-occurrence network of soil microbes underlying these shifts remain to be explored, especially for diazotrophs that contribute to the most nitrogen input in natural ecosystems. In this study, we examined how assembly processes and co-occurrence networks of soil diazotrophs changed along an elevational gradient with four elevation levels ranging from 3200 m to 3800 m using high-throughput sequencing of the nifH gene in Tibetan alpine meadows. Our analyses showed that α-diversity of soil diazotrophs was lower at low elevations (i.e., 3200 m and 3400 m) relative to high elevations (i.e., 3600 m and 3800 m). Moreover, dominant diazotrophic phyla shifted from Actinobacteria at low elevations to Proteobacteria at high elevations. The change in diazotrophic community composition along the elevation gradient was related to the differences in soil temperature, moisture, NH4+-N concentration and pH. Species rank-abundance distribution models showed that soil diazotrophic communities were mainly assembled by deterministic processes at 3200 m, 3600 m and 3800 m elevations, and by stochastic processes at the 3400 elevation. Additionally, the complexity of diazotrophic co-occurrence networks increased with increasing elevations. Collectively, our findings highlight the change in soil diazotrophic communities along the elevation gradient and the high environmental sensitivity of soil diazotrophs, and suggest that global change could have consequences for soil diazotrophic community composition and structure in montane ecosystems.

Introduction of exotic species Sonneratia apetala alters diazotrophic community and stimulates nitrogen fixation in mangrove sediments

Mangrove restoration by exotic mangrove species Sonneratia apetala has been practiced as a strategy to reconstruct mangrove wetlands. Diazotrophs in mangrove sediment are nitrogen suppliers for mangrove ecosystems. However, the response of sediment diazotrophs, especially their nitrogen fixation rates, to the introduction of exotic S. apetala remains unclear. In the present study, we compared the variations in sediment physicochemical properties, N2 fixation rate, and the composition of total bacterial and diazotrophic communities in mangrove sediments between exotic species (S. apetala) and three native species (i.e., Aegiceras corniculatum, Lumnitzera racemosa, and Rhizophora apiculata). Our results indicated that the introduction of exotic S. apetala significantly (P < 0.05) increased nutrient levels (including TC, TN, and TS) and improved the N2 fixation rate in sediments. Though S. apetala introduction had not caused significant changes in bacterial and diazotrophic alpha diversity, their community structure and composition were altered. The dominant diazotrophic groups were sulfur-oxidizing (SOB) and sulfate-reducing (SRB) bacteria. The relative abundance of Chromatiaceae (affiliated with SOB) significantly increased (P < 0.05), while the relative abundance of Desulfuromonadaceae (affiliated with SRB) decreased in S. apetala sediments. In addition, several SOB and SRB groups were identified as putative keystone taxa by network analysis, suggesting that SOB/SRB groups potentially played a central role in the biogeochemical cycles involving nitrogen (N) and sulfur (S) of the mangrove ecosystem. Redundancy analysis revealed that C/N and NH4+-N were the key factors (P < 0.05) driving sediment diazotrophic communities. This study provides new insights into the ecological impact of exotic S. apetala introduction, which has important implications for the sustainable management and restoration of mangrove ecosystems.