Diazotrophic Community Composition Research Articles

Key factors driving soil diazotrophic community assembly and nitrogenase activity in Qinghai-Tibet alpine meadows

Biological nitrogen (N) fixation by soil diazotrophs is the primary source of nitrogen for natural grasslands, especially at high altitudes such as alpine meadow ecosystems. However, the mechanisms shaping the compositions and assembly processes of diazotrophic communities, as well as soil nitrogenase activity in alpine meadows remain poorly understood. In this study, 241 soil samples were collected from alpine meadows on the Qinghai-Tibet Plateau to investigate the distribution patterns and driving factors of diazotrophic communities and nitrogenase activities. Our results showed that soil N fixation potential across all samples ranged from 28.8 to 110 nmol C2H4 g-1h-1, and Nitrospirillum and Bradyrhizobium were the most dominant diazotrophic genera. The composition and diversity of soil diazotrophic communities were mainly influenced by soil pH, and to a lesser extent by aridity index and mean annual precipitation. The abundance of nifH gene decreased linearly with soil pH, whereas the ɑ-diversity increased linearly with soil total phosphorus. Null model analysis indicated that deterministic processes governed diazotrophic community assembly, and more acidic soil conditions led to more phylogenetically clustered diazotrophic communities. Partial least squares path modeling and linear regression analyses identified diazotrophic community composition, followed by climatic factors, nifH gene abundance and total phosphorus, as the dominant regulators of soil N fixation potential in alpine meadows. Unexpectedly, soil N fixation potential was not closely related to dominant diazotrophic genera, diversity, or assembly, but highly related to the less abundant diazotrophs. This study provides a novel insight into the ecological mechanisms shaping soil biological nitrogen fixation in alpine meadow ecosystems.

Investigating drivers of free-living diazotroph activity in paddy soils across China

Microbially mediated N fixation is widespread in rice paddy ecosystems and crucial in maintaining soil fertility. However, our understanding of the factors determining the distribution of free-living diazotrophic microorganisms that perform this process in paddy fields is limited. This study investigated the spatial distribution and factors influencing presence and potential activity of free-living microorganisms capable of N2 fixation in addition to dissimilatory nitrate reduction to ammonium (DNRA), anaerobic ammonium oxidation (anammox), and denitrification in 50 paddy soils across China. Using 15N isotope tracing in laboratory incubations and microbial community analysis via metagenomics, we demonstrate that paddy soils may represent a previously underappreciated hotspot for N2 fixation with mean potential rates of 24.4 ± 17.8 nmol N g−1 h−1, 10-fold higher than DNRA (2.55 ± 0.4 nmol N g−1 h−1), and could counterbalance a portion of N2 losses through anammox and denitrification (9.24 ± 1.1 nmol N g−1 h−1). Site longitude and organic carbon (C) concentrations, as well as the diazotrophic community composition, were the dominant abiotic and biotic factors accounting for regional variations in potential N2 fixation rates. The N2 metabolic pathways predicted from the metagenome-assembled genomes (MAGs) revealed significant co-occurrence of the diazotroph marker gene nifH with denitrification-associated genes (nirS/K and nosZ) and organic C oxidation-related genes (yiaY and galM). Furthermore, enzymes involved in organic C oxidation, particularly glycoside hydrolases and glycosyltransferases, were not only phenotypically correlated with free-living N2 fixation rates but were also identified in nifH-containing MAGs, indicating the heterotrophic capabilities of diazotrophs in paddy soils. Collectively, our results underscore the substantial contribution of free-living N2 fixation to soil N fertility in paddy fields, and highlight the importance of coupling organic C oxidation with nitrate reduction to enhance N2 fixation.

Distinct Diazotrophic Communities in Water and Sediment of the Sub-Lakes in Poyang Lake, China

The sub-lakes of Poyang Lake have significant ecological value. To date, the diazotrophs in sub-lakes are unknown. Moreover, no study has simultaneously focused on diazotrophic communities in the water and sediment in natural freshwater ecosystems. In this study, we investigated the diazotrophic alpha diversity, structure, abundance, molecular ecological network, and community assembly mechanism in the water and sediment of sub-lakes using Illumina MiSeq sequencing and a quantitative polymerase chain reaction assay. The results showed that the sediment had a greater diversity of diazotrophs than the water. Proteobacteria and Spirochaetes were the dominant diazotrophic phyla in the water, whereas Proteobacteria was the dominant diazotrophic phylum in the sediment. There were significant differences in the composition of diazotrophic communities between the water and sediment. The sediment had a more complex co-occurrence network of diazotrophs than the water. Deterministic processes dominate the community assembly of diazotrophs in both the water and sediment of the sub-lakes, and the relative role of deterministic processes was stronger for sediment than water. Our study is the first to reveal the differences in the diazotrophic communities between the water and sediment in natural freshwater ecosystems and provides the fundamental scientific datasets for understanding the nitrogen fixation process in sub-lakes.

Carbon type and quantity regulate soil free-living nitrogen fixation through restructuring diazotrophic community

Free-living nitrogen fixation (FLNF) is a ubiquitous and significant source of new nitrogen in ecosystems. This process requires a substantial amount of energy, and as a result, the input of labile carbon to soils may enhance FLNF. Here, we aim to explore the precise influence of different types and quantities of carbon on FLNF. We conducted experiments where sugars, carboxylic acids, and amino acids (common labile carbon inputs via root exudates) were added to cropland and forest soils. We monitored the changes in FLNF rate and diazotrophic community in response to these additions. Our findings revealed that sugars and carboxylic acids significantly stimulated FLNF rate, with an increase of up to three orders of magnitude, while amino acids either restricted FLNF or had no effect. When a mixture of sugars and carboxylic acids was added, the stimulation of FLNF was not observed until a threshold quantity of carbon was reached, indicating competition between diazotrophs and other microbes. Furthermore, the enhanced FLNF rate following carbon addition was attributable to shifts in diazotrophic community composition (e.g., increased abundance of Paenibacillus, a genus frequently used as biofertilizers), rather than to changes in the overall abundance of diazotrophs (reflected by nifH gene copy number). Our findings highlight the significant role of carbon type and quantity in regulating soil FLNF, partly through restructuring the diazotrophic community. The results have implications for predicting the response of rhizosphere FLNF to shifts in root exudation under environmental changes and can contribute to the application of diazotrophic biofertilizers in sustainable agriculture.

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 multifrontal 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 high 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.

Endophytic diazotrophic communities from rice roots are diverse and weakly associated with soil diazotrophic community composition and soil properties.

Bacteria that promote plant growth, such as diazotrophs, are valuable tools for achieving a more sustainable production of important non-legume crops like rice. Different strategies have been used to discover new bacteria capable of promoting plant growth. This work evaluated the contribution of soil diazotrophs to the endophytic communities established in the roots of rice seedlings cultivated on seven representative soils from Uruguay. The soils were classified into two groups according to the C and clay content. qPCR, terminal restriction fragment length polymorphism (T-RFLP), and 454-pyrosequencing of the nifH gene were used for analyzing diazotrophs in soil and plantlets' roots grown from seeds of the same genotype for 25 days under controlled conditions. A similar nifH abundance was found among the seven soils, roots, or leaves. The distribution of diazotrophs was more uneven in roots than in soils, with dominance indices significantly higher than in soils (nifH T-RFLP). Dominant soils' diazotrophs were mainly affiliated to Alphaproteobacteria and Planctomycetota. Conversely, Alpha, Beta, Gammaproteobacteria, and Bacillota were predominant in different roots, though undetectable in soils. Almost no nifH sequences were shared between soils and roots. Root endophytic diazotrophs comprised a broader taxonomic range of microorganisms than diazotrophs found in soils from which the plantlets were grown and showed strong colonization patterns.

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.