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Background: The assembly of the rhizosphere community, even the diazotroph community, is mainly shaped by soil environmental factors (including soil climate and physiochemical characteristics) and plant selection. To better understand the driving forces on the active overall and nitrogen-fixing bacterial community compositions, we characterized the communities of tobacco rhizosphere soil collected from three sampling sites with a large geographic scale (> 600 km). Results: The results indicate that the diversity and community composition of the overall bacterial and diazotroph communities are obviously differed according to the sampling sites. Still, no significant difference is found between the communities in rootzone and rhizosphere samples. Climate variables including mean annual precipitation (MAP) and mean annual temperature (MAT), soil physiochemical characteristics including available nitrogen (AN), available potassium (AK) and pH are main factors that affect the bacterial and diazotroph community structures in the three sampling sites. Furthermore, MAP and MAT, AN and available phosphorus (AP), total nitrogen (TN) and organic carbon (OC), AK and electrical conductivity (EC) showed similar effects, but pH showed independent effect on the composition of the overall bacteria and diazotroph communities. However, the alpha diversity indices of active overall and nitrogen-fixing bacteria in the rhizosphere are obviously higher than in the rootzone samples, and no significant differences are observed among different sampling sites. Proteobacteria is the predominant active phylum of all samples for overall and nitrogen-fixing bacteria. Escherichia-Shigella, Achromobacter, Streptomyces and Sphingomonas are the dominant active bacterial genera, and Bradyrhizobium, Skermanella and Extensimonas are dominant active nitrogen-fixing bacteria genera in rhizosphere. Furthermore, the high active abundance of Escherichia-Shigella but low abundance of Ralstonia in all three sampling sites indicate high root-knot nematode infection and low wilt disease endemic risk. Conclusion: These results indicate that soil environmental factors contribute more to the tobacco rhizosphere bacterial community assemblage, but the rhizosphere contributes more to the diversity of active overall bacteria and nitrogen-fixing bacteria in the community. Our study provides novel knowledge for the assemble of rhizosphere bacterial and active bacteria communities across a large geographical scale. [ABSTRACT FROM AUTHOR]

Nitrous oxide (N2O) is a potent greenhouse gas and is depleting the stratospheric ozone layer. Diazotrophic N2O assimilation to biomass represents a novel biological N2O consumption pathway in addition to canonical denitrification. Thermodynamically, N2O assimilation is more favorable than dinitrogen (N2) fixation in natural environments, especially under higher N2O concentration and cooler conditions. Via isotopic tracing experiments, N2O assimilation was detected on cultured diazotrophs Crocosphaera and Trichodesmium with specific rates from 1.27 ± 0.16 × 10−4 to 2.00 ± 0.25 × 10−4 hr−1 under elevated [N2O]/[N2] conditions (0.0005–0.01) within 24‐hr incubation. The rates of N2O assimilation during the light and dark periods were statistically insignificant compared with N2 fixation activity. In a eutrophic estuary, N2O assimilation was not detected in the absence of diazotrophic activity. A competitive substrate kinetic model with experimentally calibrated parameters successfully quantified rate ratios of N2O assimilation and N2 fixation in varying substrate concentrations. The low [N2O]/[N2] ratio in natural conditions leads to N2O assimilation rate being <0.1% of N2 fixation rate, rendering negligible impact of N2O assimilation. The model was also used to predict the time required for experimental detection of N2O assimilation in isotopic tracing experiments under varying [N2O]/[N2] ratios. This study enhances the mechanistic understanding of N2O assimilation by diazotrophs, broadening the microbial nitrogen cycle by a potential N2O sink and nitrogen source for production. Plain Language Summary: Nitrous oxide (N2O) contributes to climate warming and can consume the ozone layer. In aquatic ecosystems, N2O is generally consumed only by denitrification in the absence of oxygen. Recently, nitrogen‐fixing organisms (diazotrophs) which utilize nitrogen gas (N2) to make biomass are able to assimilate N2O into biomass, demonstrating a novel N2O consumption pathway in oxygenated environments. Experimental evidence demonstrated that actively growing diazotrophs can assimilate N2O and N2 at the same time. Both observation in cultured diazotrophs and numerical model proved that the relative availabilities of N2O and N2 regulate the competition of N2O assimilation and N2 fixation. N2O assimilation is more favorable under a higher concentration ratio of N2O to N2. Model simulation could determine the time required for successful determination of N2O assimilation during experiments. Contrastingly, at high nutrient estuarine waters where diazotrophs are inactive, N2O assimilation was not detected even at high N2O concentration. Overall, N2O assimilation may be negligible in natural conditions. Key Points: Concentration ratio of N2O to N2 positively correlates with rate ratio of N2O assimilation to N2 fixationActive diazotrophs are key organism performing N2O assimilationN2O assimilation has different circadian rhythms compared with N2 fixation [ABSTRACT FROM AUTHOR]

In rice, symbiotic N2 fixation via nodule bacteroids does not take place naturally. Although N2 fixation by endophytic and associative diazotrophs has been reported in rice, the main organs and seasonal regulation for the N2 fixation have not been elucidated. In this study, seasonal changes in nitrogenase (acetylene reduction) activity and carbohydrate contents in elongated culm (EC), unelongated stem (US), and crown root (CR) were investigated in manure compost (MC)- and chemical fertilizer (CF)-applied rice. Nitrogenase activity increased after rooting (June) and reached the highest activity in US of MC-applied rice at panicle initiation (August). The sucrose content in EC continued to increase after rooting regardless of the applied materials, whereas the glucose content in US increased after rooting only in CF-applied rice, suggesting higher consumption of glucose in US of MC-applied rice. There were significant differences among bacterial microbiota in EC, US, and CR at panicle initiation. In addition, Clostridia class anaerobes were more abundant in US of MC-applied rice than in EC and CR of MC-applied rice. Such difference was not observed in US of CF-applied rice. These results suggest the suitability of US of MC-applied rice at panicle initiation as a site of N2 fixation under anaerobic conditions. [ABSTRACT FROM AUTHOR]

In rice, symbiotic N2 fixation via nodule bacteroids does not take place naturally. Although N2 fixation by endophytic and associative diazotrophs has been reported in rice, the main organs and seasonal regulation for the N2 fixation have not been elucidated. In this study, seasonal changes in nitrogenase (acetylene reduction) activity and carbohydrate contents in elongated culm (EC), unelongated stem (US), and crown root (CR) were investigated in manure compost (MC)- and chemical fertilizer (CF)-applied rice. Nitrogenase activity increased after rooting (June) and reached the highest activity in US of MC-applied rice at panicle initiation (August). The sucrose content in EC continued to increase after rooting regardless of the applied materials, whereas the glucose content in US increased after rooting only in CF-applied rice, suggesting higher consumption of glucose in US of MC-applied rice. There were significant differences among bacterial microbiota in EC, US, and CR at panicle initiation. In addition, Clostridia class anaerobes were more abundant in US of MC-applied rice than in EC and CR of MC-applied rice. Such difference was not observed in US of CF-applied rice. These results suggest the suitability of US of MC-applied rice at panicle initiation as a site of N2 fixation under anaerobic conditions.

The diazotrophic cyanobacterium Trichodesmium has been recognized as a potentially significant contributor to aerobic methane generation via several mechanisms including the utilization of methylphophonate (MPn) as a source of phosphorus. Currently, there is no information about how environmental factors regulate methane production by Trichodesmium. Here, we grew Trichodesmium IMS101 at five temperatures ranging from 16 to 31°C, and found that its methane production rates increased with rising temperatures to peak (1.028 ± 0.040 nmol CH4 μmol POC−1 day−1) at 27°C, and then declined. Its specific growth rate changed from 0.03 ± 0.01 d−1 to 0.34 ± 0.02 d−1, with the optimal growth temperature identified between 27 and 31°C. Within the tested temperature range the Q10 for the methane production rate was 4.6 ± 0.7, indicating a high sensitivity to thermal changes. In parallel, the methane production rates showed robust positive correlations with the assimilation rates of carbon, nitrogen, and phosphorus, resulting in the methane production quotients (molar ratio of carbon, nitrogen, or phosphorus assimilated to methane produced) of 227–494 for carbon, 40–128 for nitrogen, and 1.8–3.4 for phosphorus within the tested temperature range. Based on the experimental data, we estimated that the methane released from Trichodesmium can offset about 1% of its CO2 mitigation effects. [ABSTRACT FROM AUTHOR]

Crocosphaera watsonii is a unicellular N2‐fixing (diazotrophic) cyanobacterium observed in tropical and subtropical oligotrophic oceans. As a diazotroph, it can be a source of bioavailable nitrogen (N) to the microbial community in N‐limited environments, and this may fuel primary production in the regions where it occurs. Crocosphaera watsonii has been the subject of intense study, both in culture and in field populations. Here, we summarize the current understanding of the phylogenetic and physiological diversity of C. watsonii, its distribution, and its ecological niche. Analysis of the relationships among the individual Crocosphaera species and related free‐living and symbiotic lineages of diazotrophs based on the nifH gene have shown that the C. watsonii group holds a basal position and that its sequence is more similar to Rippkaea and Zehria than to other Crocosphaera species. This finding warrants further scrutiny to determine if the placement is related to a horizontal gene transfer event. Here, the nifH UCYN‐B gene copy number from a recent synthesis effort was used as a proxy for relative C. watsonii abundance to examine patterns of C. watsonii distribution as a function of environmental factors, like iron and phosphorus concentration, and complimented with a synthesis of C. watsonii physiology. Furthermore, we have summarized the current knowledge of C. watsonii with regards to N2 fixation, photosynthesis, and quantitative modeling of physiology. Because N availability can limit primary production, C. watsonii is widely recognized for its importance to carbon and N cycling in ocean ecosystems, and we conclude this review by highlighting important topics for further research on this important species. [ABSTRACT FROM AUTHOR]

A conceptual framework is proposed for understanding the relationship between temperature and biological N 2 fixation (BNF) across levels of biological organization from enzymes to ecosystems on land and in the ocean. Global compilations of measured N 2 fixation rates reveal a temperature dependence that is broadly similar in marine and terrestrial systems, including a common thermal optimum at ~25°C, suggesting an overarching enzymatic constraint. Rate–temperature relationships also reveal features unique to marine and terrestrial domains, including greater heat tolerance of terrestrial BNF, suggesting the presence of, and adaptation to, distinct thermal and ecological niches. Climate warming is projected to reduce N 2 fixation in the tropics while increasing it in higher latitudes in terrestrial and marine ecosystems, a spatial shift that would impact productivity and N cycling, unless modulated by other environmental factors and physiological adaptations. Biological N 2 fixation sustains the global inventory of nitrogenous nutrients essential for the productivity of terrestrial and marine ecosystems. Like most metabolic processes, rates of biological N 2 fixation vary strongly with temperature, making it sensitive to climate change, but a global projection across land and ocean is lacking. Here we use compilations of field and laboratory measurements to reveal a relationship between N 2 fixation rates and temperature that is similar in both domains despite large taxonomic and environmental differences. Rates of N 2 fixation increase gradually to a thermal optimum around ~25°C, and decline more rapidly toward a thermal maximum, which is lower in the ocean than on land. In both realms, the observed temperature sensitivities imply that climate warming this century could decrease N 2 fixation rates by ~50% in the tropics while increasing rates by ~50% in higher latitudes. We propose a conceptual framework for understanding the physiological and ecological mechanisms that underpin and modulate the observed temperature dependence of global N 2 fixation rates, facilitating cross-fertilization of marine and terrestrial research to assess its response to climate change. [ABSTRACT FROM AUTHOR]

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ABSTRACT Dinitrogen (N2) fixation is a crucial source of bioavailable nitrogen in carbon-dominated cold seep systems. Previous studies have shown that diazotrophy is not necessarily dependent on sulfate-dependent anaerobic oxidation of methane for energy, and diverse catabolism can fuel the high-energy-demanding process in sediments. However, it remains unclear whether diazotroph can obtain energy by sulfur oxidation in sulfur-rich cold seep water column. Here, field investigations and in situ experiments were conducted in Haima cold seep to examine the effects of diverse sources of dissolved organic matter (DOM) on N2 fixation, specifically containing sulfur, carbon, nitrogen, and phosphorus. We found that active N2 fixation occurred in the water column above the Haima cold seep, with the Dechloromonas genus dominating the diazotroph community as revealed by nifH gene using high-throughput sequencing. In situ experiments showed an increased rate of N2 fixation (1.15- to 12.70-fold compared to that in control group) and a greater relative abundance of the Dechloromonas genus following enrichment with sulfur-containing organic matter. Furthermore, metagenomic assembly and binning revealed that Dechloromonas sp. carried genes related to N2 fixation (nifDHK) and sulfur compound oxidation (fccAB and soxABCXYZ), implying that the genus potentially serves as a multifunctional mediator for N2 fixation and sulfur cycling. Our results provide new insights regarding potential coupling mechanism associated with sulfur-driven N2 fixation in methane- and sulfide-rich environments.IMPORTANCEN2 fixation is an important source of biologically available in carbon-dominated cold seep systems as little nitrogen is released by hydrocarbon seepage, thereby promoting biological productivity and the degradation of non-nitrogenous organic matter. Cold seeps are rich in diverse sources of dissolved organic matter (DOM) derived from the sinking of photosynthetic products in euphotic layer and the release of chemosynthesis products on the seafloor. However, it remains unclear whether N2 fixation is coupled to the metabolic processes of DOM, as determined by e.g., carbon, nitrogen, phosphorus, and sulfur content, for energy acquisition in sulfur-rich cold seeps. In this study, diazotroph community structure and its response to DOM compositions were revealed. Moreover, the metagenomics analysis suggested that Dechloromonas genus plays a dominant role in potential coupling N2 fixation and sulfur oxidation. Our study highlighted that sulfur oxidation in deep-sea cold seeps may serve as an energy source to drive N2 fixation.

ABSTRACT Nitrogen (N)-fixing organisms, also known as diazotrophs, play a crucial role in N-limited ecosystems by controlling the production of bioavailable N. The carbon-dominated cold-seep ecosystems are inherently N-limited, making them hotspots of N fixation. However, the knowledge of diazotrophs in cold-seep ecosystems is limited compared to other marine ecosystems. In this study, we used multi-omics to investigate the diversity and catabolism of diazotrophs in deep-sea cold-seep bottom waters. Our findings showed that the relative abundance of diazotrophs in the bacterial community reached its highest level in the cold-seep bottom waters compared to the cold-seep upper waters and non-seep bottom waters. Remarkably, more than 98% of metatranscriptomic reads aligned on diazotrophs in cold-seep bottom waters belonged to the genus Sagittula, an alphaproteobacterium. Its metagenome-assembled genome, named Seep-BW-D1, contained catalytic genes (nifHDK) for nitrogen fixation, and the nifH gene was actively transcribed in situ. Seep-BW-D1 also exhibited chemosynthetic capability to oxidize C1 compounds (methanol, formaldehyde, and formate) and thiosulfate (S2O32−). In addition, we observed abundant transcripts mapped to genes involved in the transport systems for acetate, spermidine/putrescine, and pectin oligomers, suggesting that Seep-BW-D1 can utilize organics from the intermediates synthesized by methane-oxidizing microorganisms, decaying tissues from cold-seep benthic animals, and refractory pectin derived from upper photosynthetic ecosystems. Overall, our study corroborates that carbon-dominated cold-seep bottom waters select for diazotrophs and reveals the catabolism of a novel chemosynthetic alphaproteobacterial diazotroph in cold-seep bottom waters.IMPORTANCEBioavailable nitrogen (N) is a crucial element for cellular growth and division, and its production is controlled by diazotrophs. Marine diazotrophs contribute to nearly half of the global fixed N and perform N fixation in various marine ecosystems. While previous studies mainly focused on diazotrophs in the sunlit ocean and oxygen minimum zones, recent research has recognized cold-seep ecosystems as overlooked N-fixing hotspots because the seeping fluids in cold-seep ecosystems introduce abundant bioavailable carbon but little bioavailable N, making most cold seeps inherently N-limited. With thousands of cold-seep ecosystems detected at continental margins worldwide in the past decades, the significant role of cold seeps in marine N biogeochemical cycling is emphasized. However, the diazotrophs in cold-seep bottom waters remain poorly understood. Through multi-omics, this study identified a novel alphaproteobacterial chemoheterotroph belonging to Sagittula as one of the most active diazotrophs residing in cold-seep bottom waters and revealed its catabolism.

Abstract Salinity impacts crop growth and productivity and lowers the activities of rhizosphere microbiota. The identification and utilization of habitat-specific salinity-adapted plant growth-promoting rhizobacteria (PGPR) are considered alternative strategies to improve the growth and yields of crops in salinity-affected coastal agricultural fields. In this study, we characterize strain L1I39T, the first Aquabacter species with PGPR traits isolated from a salt-tolerant pokkali rice cultivated in brackish environments. L1I39T is positive for 1-aminocyclopropane-1-carboxylate deaminase activity and nitrogen fixation and can promote pokkali rice growth by supplying fixed nitrogen under a nitrogen-deficient seawater condition. Importantly, enhanced plant growth and efficient root colonization were evident in L1I39T-inoculated plants grown under 20% seawater but not in zero-seawater conditions, identifying brackish conditions as a key local environmental factor critical for L1I39T-pokkali rice symbiosis. Detailed physiological studies revealed that L1I39T is well-adapted to brackish environments. In-depth genome analysis of L1I39T identified multiple gene systems contributing to its plant-associated lifestyle and brackish adaptations. The 16S rRNA-based metagenomic study identified L1I39T as an important rare PGPR taxon. Based on the polyphasic taxonomy analysis, we established strain L1I39T as a novel Aquabacter species and proposed Aquabacter pokkalii sp nov. Overall, this study provides a better understanding of a marine-adapted PGPR strain L1I39T that may perform a substantial role in host growth and health in nitrogen-poor brackish environments.

Salinity impacts crop growth and productivity and lowers the activities of rhizosphere microbiota. The identification and utilization of habitat-specific salinity-adapted plant growth-promoting rhizobacteria (PGPR) are considered alternative strategies to improve the growth and yields of crops in salinity-affected coastal agricultural fields. In this study, we characterize strain L1I39T, the first Aquabacter species with PGPR traits isolated from a salt-tolerant pokkali rice cultivated in brackish environments. L1I39T is positive for 1-aminocyclopropane-1-carboxylate deaminase activity and nitrogen fixation and can promote pokkali rice growth by supplying fixed nitrogen under a nitrogen-deficient seawater condition. Importantly, enhanced plant growth and efficient root colonization were evident in L1I39T-inoculated plants grown under 20% seawater but not in zero-seawater conditions, identifying brackish conditions as a key local environmental factor critical for L1I39T-pokkali rice symbiosis. Detailed physiological studies revealed that L1I39T is well-adapted to brackish environments. In-depth genome analysis of L1I39T identified multiple gene systems contributing to its plant-associated lifestyle and brackish adaptations. The 16S rRNA-based metagenomic study identified L1I39T as an important rare PGPR taxon. Based on the polyphasic taxonomy analysis, we established strain L1I39T as a novel Aquabacter species and proposed Aquabacter pokkalii sp nov. Overall, this study provides a better understanding of a marine-adapted PGPR strain L1I39T that may perform a substantial role in host growth and health in nitrogen-poor brackish environments. [ABSTRACT FROM AUTHOR]

The crucial functional arbuscular mycorrhizal fungi (AMF) and diazotrophs play pivotal roles in nutrient cycling during vegetation restoration. However, the impact of managed vegetation restoration strategies on AMF and diazotroph communities remains unclear. In this study, we investigated the community structure and diversity of AMF and diazotrophs in a karst region undergoing managed vegetation restoration from cropland. Soil samples were collected from soils under three vegetation restoration strategies, plantation forest (PF), forage grass (FG), and a mixture of plantation forest and forage grass (FF), along with a control for cropland rotation (CR). The diversity of both AMF and diazotrophs was impacted by managed vegetation restoration. Specifically, the AMF Shannon index was higher in CR and PF compared to FF. Conversely, diazotroph richness was lower in CR, PF, and FG than in FF. Furthermore, both AMF and diazotroph community compositions differed between CR and FF. The relative abundance of AMF taxa, such as Glomus, was lower in FF compared to the other three land-use types, while Racocetra showed the opposite trend. Among diazotroph taxa, the relative abundance of Anabaena, Nostoc, and Rhizobium was higher in FF than in CR. Soil properties such as total potassium, available potassium, pH, and total nitrogen were identified as the main factors influencing AMF and diazotroph diversity. These findings suggest that AMF and diazotroph communities were more sensitive to FF rather than PF and FG after managed vegetation restoration from cropland, despite similar levels of soil nutrients among PF, FG, and FF. Consequently, the integration of diverse economic tree species and forage grasses in mixed plantations notably altered the diversity and species composition of AMF and diazotrophs, primarily through the promotion of biocrust formation and root establishment. [ABSTRACT FROM AUTHOR]

Plant breeding and genetics play a major role in the adaptation of plants to meet human needs. The current requirement to make agriculture more sustainable can be partly met by a greater reliance on biological nitrogen fixation by symbiotic diazotrophic microorganisms that provide crop plants with ammonium. Select accessions of the cereal crop sorghum (Sorghum bicolor (L.) Moench) form mucilage-producing aerial roots that harbor nitrogen-fixing bacteria. Breeding programs aimed at developing sorghum varieties that support diazotrophs will benefit from a detailed understanding of the genetic and environmental factors contributing to aerial root formation. A genome-wide association study of the sorghum minicore, a collection of 242 landraces, and 30 accessions from the sorghum association panel was conducted in Florida and Wisconsin and under 2 fertilizer treatments to identify loci associated with the number of nodes with aerial roots and aerial root diameter. Sequence variation in genes encoding transcription factors that control phytohormone signaling and root system architecture showed significant associations with these traits. In addition, the location had a significant effect on the phenotypes. Concurrently, we developed F2 populations from crosses between bioenergy sorghums and a landrace that produced extensive aerial roots to evaluate the mode of inheritance of the loci identified by the genome-wide association study. Furthermore, the mucilage collected from aerial roots contained polysaccharides rich in galactose, arabinose, and fucose, whose composition displayed minimal variation among 10 genotypes and 2 fertilizer treatments. These combined results support the development of sorghums with the ability to acquire nitrogen via biological nitrogen fixation. [ABSTRACT FROM AUTHOR]

Iron (Fe) is an essential micronutrient for phytoplankton growth, but its scarcity in seawater limits primary productivity across much of the ocean. Most dissolved Fe (DFe) in seawater is complexed with Fe-binding organic ligands, a poorly constrained fraction of dissolved organic matter (DOM), which increase Fe residence time and impact Fe bioavailability. Here, we present the conditional concentration (LFe) and binding-strength (log KcondFe'L) of Fe-binding ligands in the Western Tropical South Pacific (WTSP) Ocean during the GEOTRACES TONGA cruise (GPpr14). The transect crossed the Lau basin, a region subject to shallow hydrothermal Fe inputs that fuel intense diazotrophic activity, the oligotrophic South Pacific gyre, and the Melanesian basin. Organic speciation was analyzed by competitive ligand exchange adsorptive cathodic stripping voltammetry (CLE-AdCSV) using salicylaldoxime at 25 µM. We found a high mean LFe of 5.2 ± 1.2 nMeqFe (n = 103) across the entire transect, predominantly consisting of intermediate strength L2 ligands (84%; mean log KcondFe'L of 11.6 ± 0.4), consistent with humic-like substances. DFe correlated with the humic-like component of the fluorescent DOM (HS-like FDOM), yet the electroactive Fe-binding humic-like substances (LFeHS) accounted for only 20 ± 13% of LFe in the mixed layer and 8 ± 6% in deep waters. Ligands were in large excess compared to DFe (mean excess ligand eLFe = 4.6 ± 1.1 nMeqFe), suggesting poor stabilization of DFe inputs. High LFe (up to 9 nMeqFe) in samples close to hydrothermal sites could be due to detoxification strategies from plankton communities toward hydrothermally-fueled toxic trace metals other than Fe, with an apparent dilution of the DOM from the Lau basin into neighboring regions. We also observed a different peak potential of the Fe salicylaldoxime complex detected by CLE-AdCSV between the Lau and Melanesian basins, and between surface and deep waters. To our knowledge, this change in potential has not previously been reported; whether this represents a novel detection of specificities in DOM composition merits further investigation. Competition between Fe and competing metals for ligand binding sites could favor DFe oxidation and precipitation near hydrothermal vents and explain the absence of strong Fe stabilization in the WTSP. [ABSTRACT FROM AUTHOR]

The establishment of eco-friendly principles in agriculture requires, from one side, a reduced dependence on chemical fertilizers, pesticides and herbicides, and on the other, a maintenance of consistent yield and high plant productivity. Such substantial shift in the contemporary agricultural practices can be achieved only if a wide variety of alternative options is available. Furthermore, in order to become part of agricultural practices these options have to be affordable in a broad scale and effective against the loss of biodiversity and reduced soil fertility. The current study presents data about the metabolic activity and microbial community` structure in two soil samples – one treated with chemical fertilizer and another treated with chemical fertilizer reduced by 25% dose and supplemented with commercially available biofertilizer. Metabolic activity and properties of microbial soil communities were assessed by the BIOLOG® EcoPlate technique. The estimated parameters included average well)color development (AWCD) and utilisation of six guilds of substrates. Several functional indexes that are used for evaluation of community diversity and evenness were also estimated. The AWCD, expressed either as total activity or as guilds utilisation during the EcoPlate incubation period of seven days did not reveal significant difference between variants. However, most of the estimated functional indexes consistently indicated higher microbial diversity and more balanced structure of microbial community in the biofertilizer-supplemented variant. The results clearly implied that the biofertilizer based on nitrogen-fixing bacteria can positively influence the structure of soil communities and its application could be beneficial for soil fertility and plant productivity. [ABSTRACT FROM AUTHOR]

Maize (Zea mays) requires substantial amounts of nitrogen, posing a challenge for its cultivation. Recent work discovered that some ancient Mexican maize landraces harbored diazotrophic bacteria in mucilage secreted by their aerial roots. To see if this trait is retained in modern maize, we conducted a field study of aerial root mucilage (ARM) in 258 inbred lines. We observed that ARM secretion is common in modern maize, but the amount significantly varies, and only a few lines have retained the nitrogen‐fixing traits found in ancient landraces. The mucilage of the high‐ARM inbred line HN5‐724 had high nitrogen‐fixing enzyme activity and abundant diazotrophic bacteria. Our genome‐wide association study identified 17 candidate genes associated with ARM across three environments. Knockouts of one candidate gene, the subtilase family gene ZmSBT3, confirmed that it negatively regulates ARM secretion. Notably, the ZmSBT3 knockout lines had increased biomass and total nitrogen accumulation under nitrogen‐free culture conditions. High ARM was associated with three ZmSBT3 haplotypes that were gradually lost during maize domestication, being retained in only a few modern inbred lines such as HN5‐724. In summary, our results identify ZmSBT3 as a potential tool for enhancing ARM, and thus nitrogen fixation, in maize. [ABSTRACT FROM AUTHOR]

Di-nitrogen (N2) fixation rates, and the diversity of the organisms that fix N2, remain largely unconstrained in the aquatic landscapes or aquascapes (e.g., lakes, wetlands, streams, rivers, estuaries) between land and sea. As a result, we lack a mechanistic understanding of the controls and contributions of N2 fixation across disparate aquatic environments, and cannot accurately incorporate this process into local and global nitrogen (N) budgets. This special issue brings together papers highlighting current advances in understanding of N2 fixation within and across all aquatic habitats, integrating novel methodology for studying N2 fixation, quantification of N2 fixation fluxes in understudied habitats, the role of N2 fixation in biotic assemblages, and the rate and fate of fixed N in heterogeneous landscapes. Together, these papers address important gaps in understanding and highlight the frontiers of research on N2 fixation in aquatic habitats. [ABSTRACT FROM AUTHOR]

Subtropical‐subpolar transition water is a potential domain for N2 fixation, but the understanding of N2 fixation in such waters remains incomplete. We simultaneously examined the N2 fixation activity and community structures of diazotrophs and all prokaryotes from the surface to just above the seafloor off Patagonia in the transitional region of the eastern South Pacific Ocean. N2 fixation activity was not detected in the surface waters, but was observed sporadically and only in subpolar bathypelagic waters (>1,000 m) at very low rates (0.02–0.06 nmol N L−1 d−1). By contrast, the nifH gene, a key gene involved in N2 fixation, was detected widely from the surface to the bottom waters. The majority of diazotrophs were classified as non‐cyanobacterial diazotrophs (NCDs), and the nifH amino acid sequences of major diazotrophs were similar to sequences detected in the Southern Ocean, the aphotic zone and sediment of other oceans, and estuarine waters, suggesting that the NCDs are distributed across diverse marine environments. The overall prokaryotic communities were generally similar to those in other open ocean regions at the phylum level (class level for Proteobacteria) and differed among water depths. Diazotrophs, in contrast, showed vertical and horizontal heterogeneity below the euphotic zone and little association with water depth, indicating a lack of cohesion within the community, which may characterize diazotroph community in the transitional surface water and aphotic zones. Elucidating this community heterogeneity may provide pivotal information about N2 fixation in these waters. Plain Language Summary: Marine N2 fixation is the main source of nitrogen to the oceans but was generally considered a local process that occurs primarily in the surface layers of tropical and subtropical oligotrophic waters. However, recent studies shows that N2 fixation occurs in diverse environments, including polar regions. The subtropical‐subpolar transition region has recently been recognized as a region where N2 fixation occurs, but studies of diazotrophy in this region have only just begun and information remains incomplete. In this study, diazotrophy in the transition region off Patagonia was examined throughout the water column. N2 fixation activity was sporadic in the deep waters of this region. By contrast, diazotrophs were detected from the surface to just above the seafloor. Unlike other prokaryotic communities, diazotrophic communities had heterogenous vertical and horizontal distribution below the euphotic zone. The heterogeneity of diazotrophs may be key to understanding N2 fixation in transitional surface and aphotic waters. Key Points: N2 fixation activity was detected sporadically and only in subpolar bathypelagic waters off western PatagoniaDiazotrophs were nearly omnipresent from the surface to just above the seafloorIn contrast to other prokaryotes, the vertical and horizontal distributions of diazotrophs were heterogeneous [ABSTRACT FROM AUTHOR]

Phyllosphere exudates create specialized microhabitats that shape microbial community diversity. We explored the microbiome associated with two sorghum phyllosphere exudates, the epicuticular wax and aerial root mucilage. We assessed the microbiome associated with the wax from sorghum plants over two growth stages, and the root mucilage additionally from nitrogen-fertilized and nonfertilized plants. In parallel, we isolated and characterized hundreds of bacteria from wax and mucilage, and integrated data from cultivation-independent and cultivation-dependent approaches to gain insights into exudate diversity and bacterial phenotypes. We found that Sphingomonadaceae and Rhizobiaceae families were the major taxa in the wax regardless of water availability and plant developmental stage to plants. The cultivation-independent mucilage-associated bacterial microbiome contained the families Erwiniaceae, Flavobacteriaceae, Rhizobiaceae, Pseudomonadaceae, and Sphingomonadaceae, and its structure was strongly influenced by sorghum development but only modestly influenced by fertilization. In contrast, the fungal community structure of mucilage was strongly affected by the year of sampling but not by fertilization or plant developmental stage, suggesting a decoupling of fungal–bacterial dynamics in the mucilage. Our bacterial isolate collection from wax and mucilage had several isolates that matched 100% to detected amplicon sequence variants, and were enriched on media that selected for phenotypes that included phosphate solubilization, putative diazotrophy, resistance to desiccation, capability to grow on methanol as a carbon source, and ability to grow in the presence of linalool and β-caryophyllene (terpenes in sorghum wax). This work expands our understanding of the microbiome of phyllosphere exudates and supports our long-term goal to translate microbiome research to support sorghum cultivation.

Abstract Background A 2-year field trial was conducted to test the effect of biochar addition (0, 15, 30, and 45 t hm−2) on soil properties, nutrients, diazotrophic community diversity, abundance, and structure, and soybean growth, yield, and quality. Furthermore, we aimed to explore the responses of diazotrophs, grain yield, and quality to nine soil environmental factors. Rhizosphere soil and plant samples were collected after harvest. Results Biochar application resulted in a lower soil bulk density (γ d) but higher total organic carbon (TOC), effective phosphorus (AP) and total nitrogen (TN). Compared with untreated soil, the diversity index of diazotrophic bacteria in biochar-amended soil decreased, but the abundance of diazotrophic bacteria increased. The microbial community remained stable when a small amount of biochar was applied but changed as biochar amount increased. Furthermore, biochar reduced the proportion of unique nitrogen-fixing bacteria, but did not affect that of common nitrogen-fixing bacteria between biochar-amended and untreated soils, and increased the relative abundance of Bradyrhizobium (B9 vs. B0) and Sinorhizobium (B18 or B21 vs. B0) involved in symbiotic nitrogen fixation. The main components and content of fatty acids (except for stearic acid) and the content of protein and soybean oil remained stable under biochar application. The low biochar treatment (15 t hm−2) promoted soybean growth and yield. Redundancy analysis suggested that TN greatly influenced the diazotrophic community structure at the phylum and genus levels, and that pH, TOC, and NO3 −-N greatly influenced grain yield and quality. Conclusions Soil diazotroph environment can be improved by targeted farmland implementation based on changes in soil physicochemical properties, which would benefit biological N fixation in agricultural soils and further increase economic benefit. Graphical Abstract

Iron (Fe) is an essential micronutrient for phytoplankton growth, but its scarcity in seawater limits primary productivity across much of the ocean. Most dissolved Fe (DFe) in seawater is complexed with Fe-binding organic ligands, a poorly constrained fraction of dissolved organic matter (DOM), which increase Fe residence time and impact Fe bioavailability. Here, we present the conditional concentration (LFe) and binding-strength (log KFe'Lcond) of Fe-binding ligands in the Western Tropical South Pacific (WTSP) Ocean during the GEOTRACES TONGA cruise (GPpr14). The transect crossed the Lau basin, a region subject to shallow hydrothermal Fe inputs that fuel intense diazotrophic activity, the oligotrophic South Pacific gyre, and the Melanesian basin. Organic speciation was analyzed by competitive ligand exchange adsorptive cathodic stripping voltammetry (CLE-AdCSV) using salicylaldoxime at 25 µM. We found a high mean LFe of 5.2 ± 1.2 nMeqFe (n = 103) across the entire transect, predominantly consisting of intermediate strength L2 ligands (84%; mean log KFe'Lcond of 11.6 ± 0.4), consistent with humic-like substances. DFe correlated with the humic-like component of the fluorescent DOM (HS-like FDOM), yet the electroactive Fe-binding humic-like substances (LFeHS) accounted for only 20 ± 13% of LFe in the mixed layer and 8 ± 6% in deep waters. Ligands were in large excess compared to DFe (mean excess ligand eLFe = 4.6 ± 1.1 nMeqFe), suggesting poor stabilization of DFe inputs. High LFe (up to 9 nMeqFe) in samples close to hydrothermal sites could be due to detoxification strategies from plankton communities toward hydrothermally-fueled toxic trace metals other than Fe, with an apparent dilution of the DOM from the Lau basin into neighboring regions. We also observed a different peak potential of the Fe salicylaldoxime complex detected by CLE-AdCSV between the Lau and Melanesian basins, and between surface and deep waters. To our knowledge, this change in potential has not previously been reported; whether this represents a novel detection of specificities in DOM composition merits further investigation. Competition between Fe and competing metals for ligand binding sites could favor DFe oxidation and precipitation near hydrothermal vents and explain the absence of strong Fe stabilization in the WTSP.

Abstract Background Plants sustain intimate relationships with diverse microbes. It is well-recognized that these plant-associated microbiota shape individual performance and fitness of host plants, but much remains to be explored regarding how they exert their function and maintain their homeostasis. Results Here, using pink lady (Heterotis rotundifolia) as a study plant, we investigated the phenomenon of microbiota-mediated nitrogen fixation and elucidated how this process is steadily maintained in the root mucilage microhabitat. Metabolite and microbiota profiling showed that the aerial root mucilage is enriched in carbohydrates and diazotrophic bacteria. Nitrogen isotope-labeling experiments, 15N natural abundance, and gene expression analysis indicated that the aerial root-mucilage microbiota could fix atmospheric nitrogen to support plant growth. While the aerial root mucilage is a hotspot of nutrients, we did not observe high abundance of other environmental and pathogenic microbes inside. We further identified a fungus isolate in mucilage that has shown broad-spectrum antimicrobial activities, but solely allows the growth of diazotrophic bacteria. This “friendly” fungus may be the key driver to maintain nitrogen fixation function in the mucilage microhabitat. Video Abstract Conclusion The discovery of new biological function and mucilage-habitat friendly fungi provides insights into microbial homeostasis maintenance of microenvironmental function and rhizosphere ecology.

Endophytic nitrogen-fixing (diazotrophic) bacteria are essential members of the microbiome of switchgrass (Panicum virgatum), considered to be an important commodity crop in bioenergy production. While endophytic diazotrophs are known to provide fixed atmospheric nitrogen to their host plant, there are many other plant growth-promoting (PGP) capabilities of these organisms to be demonstrated. The diversity of PGP traits across different taxa of switchgrass-colonizing endophytes is understudied, yet critical for understanding endophytic function and improving cultivation methods of important commodity crops. Here, we present the isolation and characterization of three diazotrophic endophytes: Azospirillum agricola R1C, Klebsiella variicola F10Cl, and Raoultella terrigena R1Gly. Strains R1C and F10Cl were isolated from switchgrass and strain R1Gly, while isolated from tobacco, is demonstrated herein to colonize switchgrass. Each strain exhibited highly diverse genomic and phenotypic PGP capabilities. Strain F10Cl and R1Gly demonstrated the highest functional similarity, suggesting that, while endophyte community structure may vary widely based on host species, differences in functional diversity are not a clearly delineated. The results of this study advance our understanding of diazotrophic endophyte diversity, which will allow us to design robust strategies to improve cultivation methods of many economically important commodity crops.

The crucial functional arbuscular mycorrhizal fungi (AMF) and diazotrophs play pivotal roles in nutrient cycling during vegetation restoration. However, the impact of managed vegetation restoration strategies on AMF and diazotroph communities remains unclear. In this study, we investigated the community structure and diversity of AMF and diazotrophs in a karst region undergoing managed vegetation restoration from cropland. Soil samples were collected from soils under three vegetation restoration strategies, plantation forest (PF), forage grass (FG), and a mixture of plantation forest and forage grass (FF), along with a control for cropland rotation (CR). The diversity of both AMF and diazotrophs was impacted by managed vegetation restoration. Specifically, the AMF Shannon index was higher in CR and PF compared to FF. Conversely, diazotroph richness was lower in CR, PF, and FG than in FF. Furthermore, both AMF and diazotroph community compositions differed between CR and FF. The relative abundance of AMF taxa, such as Glomus, was lower in FF compared to the other three land-use types, while Racocetra showed the opposite trend. Among diazotroph taxa, the relative abundance of Anabaena, Nostoc, and Rhizobium was higher in FF than in CR. Soil properties such as total potassium, available potassium, pH, and total nitrogen were identified as the main factors influencing AMF and diazotroph diversity. These findings suggest that AMF and diazotroph communities were more sensitive to FF rather than PF and FG after managed vegetation restoration from cropland, despite similar levels of soil nutrients among PF, FG, and FF. Consequently, the integration of diverse economic tree species and forage grasses in mixed plantations notably altered the diversity and species composition of AMF and diazotrophs, primarily through the promotion of biocrust formation and root establishment.

The genus Herbaspirillum gained the spotlight due to the several reports of diazotrophic strains and promising results in plant-growth field assays. However, as diversity exploration of Herbaspirillum species gained momentum, it became clearer that the plant beneficial lifestyle was not the only form of ecological interaction in this genus, due to reports of phytopathogenesis and nosocomial infections. Here we performed a deep search across all publicly available Herbaspirillum genomes. Using a robust core genome phylogeny, we have found that all described species are well delineated, being the only exception H. aquaticum and H. huttiense clade. We also uncovered that the nif genes are only highly prevalent in H. rubrisubalbicans; however, irrespective to the species, all nif genes share the same gene arrangement with high protein identity, and are present in only two main types, in inverted strands. By means of a NifHDKENB phylogenetic tree, we have further revealed that the Herbaspirillum nif sequences may have been acquired from the same last common ancestor belonging to the Nitrosomonadales order. [ABSTRACT FROM AUTHOR]

Microalgae are used in various biotechnological processes, such as biofuel production due to their high biomass yields, agriculture as biofertilizers, production of high-value-added products, decontamination of wastewater, or as biological models for carbon sequestration. The number of these biotechnological applications is increasing, and as such, any advances that contribute to reducing costs and increasing economic profitability can have a significant impact. Nitrogen fixing organisms, often called diazotroph, also have great biotechnological potential, mainly in agriculture as an alternative to chemical fertilizers. Microbial consortia typically perform more complex tasks than monocultures and can execute functions that are challenging or even impossible for individual strains or species. Interestingly, microalgae and diazotrophic organisms are capable to embrace different types of symbiotic associations. Certain corals and lichens exhibit this symbiotic relationship in nature, which enhances their fitness. However, this relationship can also be artificially created in laboratory conditions with the objective of enhancing some of the biotechnological processes that each organism carries out independently. As a result, the utilization of microalgae and diazotrophic organisms in consortia is garnering significant interest as a potential alternative for reducing production costs and increasing yields of microalgae biomass, as well as for producing derived products and serving biotechnological purposes. This review makes an effort to examine the associations of microalgae and diazotrophic organisms, with the aim of highlighting the potential of these associations in improving various biotechnological processes. [ABSTRACT FROM AUTHOR]

Background: A 2-year field trial was conducted to test the effect of biochar addition (0, 15, 30, and 45 t hm−2) on soil properties, nutrients, diazotrophic community diversity, abundance, and structure, and soybean growth, yield, and quality. Furthermore, we aimed to explore the responses of diazotrophs, grain yield, and quality to nine soil environmental factors. Rhizosphere soil and plant samples were collected after harvest. Results: Biochar application resulted in a lower soil bulk density (γd) but higher total organic carbon (TOC), effective phosphorus (AP) and total nitrogen (TN). Compared with untreated soil, the diversity index of diazotrophic bacteria in biochar-amended soil decreased, but the abundance of diazotrophic bacteria increased. The microbial community remained stable when a small amount of biochar was applied but changed as biochar amount increased. Furthermore, biochar reduced the proportion of unique nitrogen-fixing bacteria, but did not affect that of common nitrogen-fixing bacteria between biochar-amended and untreated soils, and increased the relative abundance of Bradyrhizobium (B9 vs. B0) and Sinorhizobium (B18 or B21 vs. B0) involved in symbiotic nitrogen fixation. The main components and content of fatty acids (except for stearic acid) and the content of protein and soybean oil remained stable under biochar application. The low biochar treatment (15 t hm−2) promoted soybean growth and yield. Redundancy analysis suggested that TN greatly influenced the diazotrophic community structure at the phylum and genus levels, and that pH, TOC, and NO3−-N greatly influenced grain yield and quality. Conclusions: Soil diazotroph environment can be improved by targeted farmland implementation based on changes in soil physicochemical properties, which would benefit biological N fixation in agricultural soils and further increase economic benefit. [ABSTRACT FROM AUTHOR]

Peat mosses (Sphagnum spp.) are keystone species in boreal peatlands, where they dominate net primary productivity and facilitate the accumulation of carbon in thick peat deposits. Sphagnum mosses harbor a diverse assemblage of microbial partners, including N2‐fixing (diazotrophic) and CH4‐oxidizing (methanotrophic) taxa that support ecosystem function by regulating transformations of carbon and nitrogen. Here, we investigate the response of the Sphagnum phytobiome (plant + constituent microbiome + environment) to a gradient of experimental warming (+0°C to +9°C) and elevated CO2 (+500 ppm) in an ombrotrophic peatland in northern Minnesota (USA). By tracking changes in carbon (CH4, CO2) and nitrogen (NH4‐N) cycling from the belowground environment up to Sphagnum and its associated microbiome, we identified a series of cascading impacts to the Sphagnum phytobiome triggered by warming and elevated CO2. Under ambient CO2, warming increased plant‐available NH4‐N in surface peat, excess N accumulated in Sphagnum tissue, and N2 fixation activity decreased. Elevated CO2 offset the effects of warming, disrupting the accumulation of N in peat and Sphagnum tissue. Methane concentrations in porewater increased with warming irrespective of CO2 treatment, resulting in a ~10× rise in methanotrophic activity within Sphagnum from the +9°C enclosures. Warming's divergent impacts on diazotrophy and methanotrophy caused these processes to become decoupled at warmer temperatures, as evidenced by declining rates of methane‐induced N2 fixation and significant losses of keystone microbial taxa. In addition to changes in the Sphagnum microbiome, we observed ~94% mortality of Sphagnum between the +0°C and +9°C treatments, possibly due to the interactive effects of warming on N‐availability and competition from vascular plant species. Collectively, these results highlight the vulnerability of the Sphagnum phytobiome to rising temperatures and atmospheric CO2 concentrations, with significant implications for carbon and nitrogen cycling in boreal peatlands. [ABSTRACT FROM AUTHOR]

In the subtropical North Pacific, the east‐west gradient of iron deposition as atmospheric Asian dust strongly affects the zonal distribution of biological N2 fixation activity in numerical models, but the in‐situ relationship at a basin‐scale is not well examined. We examined the trans‐Pacific zonal variation in N2 fixation activity on 23°N in summer along with environmental parameters that potentially influence diazotrophy. Dissolved inorganic iron (DFe) was consistently low (<0.4 nM) throughout the transect. The atmospheric dust iron (dust‐Fe) flux increased westward, whereas phosphate and labile phosphoric monoesters in the surface decreased westward. N2 fixation varied between 34.6 and 298 μmol‐N m−2 d−1 and was high (>200 μmol‐N m−2 d−1) in the central area (150–180°W). N2 fixation rates significantly increased with dust‐Fe input only in the western area (137–180°E), whereas the contribution of DFe diffused from below the euphotic zone was often larger in the eastern area (120–170°W). N2 fixation was considerably low in the phosphate‐depleted western area despite the excess amount of iron relative to phosphate, and it increased with the labile phosphoric monoesters stock. These indicated that N2 fixation was primarily limited by phosphorus in the western area, though this activity also increased with iron supply from dust, likely due to phosphorus‐iron co‐limitation. In contrast, in the phosphorus‐repleted eastern area, iron supplied from dust and below the euphotic zone appeared to limit N2 fixation. Overall, N2 fixation in the subtropical North Pacific was likely limited by zonally different factors relating to iron and phosphorus availability. Plain Language Summary: N2 fixation, a process that converts N2 gas into ammonia, substantially affects biological production in subtropical ecosystems, where nitrogenous nutrients are scarce. The availability of both iron and phosphorus are primary factors controlling the growth of N2‐fixing organisms. In North Pacific subtropical waters, iron input as atmospheric Asian dust is considered to control the east‐west variation in N2 fixation; however, their relationship is not well‐examined. Here, we investigated the trans‐Pacific distribution of N2 fixation activity with iron and phosphorus availability. Our results show that dust input correlates with N2 fixation in the western area (137–180°E), which is in contrast with the eastern area (120–170°W), where vertically diffused iron was also important. In the phosphate‐depleted western area, N2 fixation positively correlated with the proxies of phosphorus supply, suggesting phosphorus limitation. This indicates phosphorus availability substantially controls the zonal distribution of N2 fixation in the subtropical North Pacific through co‐limitation with iron. The limitations are relaxed in the central area (150–180°W), likely due to elevated nutrient supply caused by bottom topography, resulting in a peak in N2 fixation. Therefore, the dynamics of both iron and phosphorus must be considered simultaneously to predict the response of N2 fixation to environmental changes. Key Points: Along the zonal transect in the subtropical North Pacific, N2 fixation activity was high in the central area (150–180°W)The central North Pacific was characterized by intermediate iron and phosphorus supply in the euphotic zoneEast‐west gradient of phosphorus availability controls the N2 fixation in the North Pacific through likely co‐limitation with iron [ABSTRACT FROM AUTHOR]

Considering the crucial role of soil diazotrophs and root arbuscular mycorrhizal fungi (AMF) in soil nutrient cycling during ecosystem restoration, diazotroph and AMF communities may be determined by slope position. However, the effect of slope position on diazotroph and AMF abundance, diversity, and community composition of karst ecosystems remains unknown. In this study, soil diazotrophs and root AMF characteristics on varying slope positions were assessed in a karst shrub ecosystem. The results displayed that the abundance of soil diazotrophs and root AMF diversity were significantly affected by slope position. Diazotroph abundance accompanied by soil nutrient and plant richness was higher on the lower slopes than the upper slopes, whereas root AMF diversity displayed the opposite trend. The soil diazotroph and root AMF community composition differed among the upper, middle, and lower slopes. The dominant taxa of soil diazotrophs and root AMF at the order level were Rhizobiales and Glomerales, respectively. Moreover, the diazotroph order of Nostocales and the AMF order of Paraglomerales were richer on the upper slopes than on the lower slopes. The slope position directly affected the plant diversity and soil nutrient distribution, indirectly affecting the diazotroph and AMF communities. Increased available nitrogen on the lower slope caused great diazotroph abundance by stimulating plant growth with sufficient carbohydrates. However, low soil nutrients and plant diversity but high plant root biomass induced more root AMF diversity on the upper slope than on the lower slope. Therefore, this study expands the knowledge of soil diazotroph and root AMF ecological functions along different slope positions during vegetation recovery for the successive stages of grass and shrub in the karst region. [ABSTRACT FROM AUTHOR]

Abstract Background The importance of symbiosis has long been recognized on coral reefs, where the photosynthetic dinoflagellates of corals (Symbiodiniaceae) are the primary symbiont. Numerous studies have now shown that a diverse assemblage of prokaryotes also make-up part of the microbiome of corals. A subset of these prokaryotes is capable of fixing nitrogen, known as diazotrophs, and is also present in the microbiome of scleractinian corals where they have been shown to supplement the holobiont nitrogen budget. Here, an analysis of the microbiomes of 16 coral species collected from Australia, Curaçao, and Hawai’i using three different marker genes (16S rRNA, nifH, and ITS2) is presented. These data were used to examine the effects of biogeography, coral traits, and ecological life history characteristics on the composition and diversity of the microbiome in corals and their diazotrophic communities. Results The prokaryotic microbiome community composition (i.e., beta diversity) based on the 16S rRNA gene varied between sites and ecological life history characteristics, but coral morphology was the most significant factor affecting the microbiome of the corals studied. For 15 of the corals studied, only two species Pocillopora acuta and Seriotopora hystrix, both brooders, showed a weak relationship between the 16S rRNA gene community structure and the diazotrophic members of the microbiome using the nifH marker gene, suggesting that many corals support a microbiome with diazotrophic capabilities. The order Rhizobiales, a taxon that contains primarily diazotrophs, are common members of the coral microbiome and were eight times greater in relative abundances in Hawai’i compared to corals from either Curacao or Australia. However, for the diazotrophic component of the coral microbiome, only host species significantly influenced the composition and diversity of the community. Conclusions The roles and interactions between members of the coral holobiont are still not well understood, especially critical functions provided by the coral microbiome (e.g., nitrogen fixation), and the variation of these functions across species. The findings presented here show the significant effect of morphology, a coral “super trait,” on the overall community structure of the microbiome in corals and that there is a strong association of the diazotrophic community within the microbiome of corals. However, the underlying coral traits linking the effects of host species on diazotrophic communities remain unknown. Video Abstract

Coral reefs are known for being highly productive ecosystems in oligotrophic oceans, which is commonly referred to as the Darwin’s Paradox. Nitrogen is an essential component of organisms, but it limits primary productivity in most euphotic ocean, including the coral reef system. Therefore, understanding nitrogen’s transfer and transformation within the coral holobiont is essential to comprehend the holobiont homeostasis and functioning mechanisms, which may help to explain the Darwin’s Paradox. Previous studies have pointed out the fundamental importance of nitrogen cycling between coral host and symbiotic algae. Recently, increasing researches, particularly in quantitative aspect, have significantly improved our understandings of the various roles of nitrogen pathways in regulating the inter-relationship among coral host and symbiotic algae and the associated microbiome. In this paper, we synthesized knowledge advances of different nitrogen processes in coral holobionts standing on the nitrogen cycle perspective. We extracted consensus and contradictions from published research results regarding nitrogen flows of coral holobiont. This review presented the temporal and spatial variation of nitrogen fixation and analyzed the global nitrogen processes rates in coral holobionts. We also summarized projections of specific nitrogen processes of coral holobionts facing climate change from limited reports. We realized that there are significant gaps in our understanding of nitrogen processes in coral holobionts, which hindering our comprehension of nitrogen balance in coral holobionts and, therefore, the coral reef systems. These gaps include the roles and relative importance of nitrification, denitrification, and DNRA in coral holobionts, as well as the self-regulation mechanisms to maintain nitrogen-homeostasis in short-term and long-term, particularly in the context of environmental changes. At the end, we provide our opinions on research methods regarding quantitative coral research in the future.

We examined the response of microbial communities in the model sea anemone Exaiptasia diaphana (Aiptasia) to short-term thermal elevation. Through 16S rRNA gene sequencing, we characterized the microbiomes of symbiotic (with algal symbionts) and aposymbiotic (bleached) anemones under ambient (27°C) and heat-stressed (34°C) conditions for 8-10 days, using both replicated endpoint and non-replicated time-course approaches. Consistent with prior studies, we observed a stable abundance of bacteria from the families Alteromonadaceae and Rhodobacteraceae, though with wide variation among individual anemones. We observed that symbiotic state conferred a larger impact on the microbiome than heat stress, implying the microbiome may play a metabolic role in the maintenance of cnidarian-dinoflagellate symbiosis. In particular, Pelobacter, an anaerobic sulfate reducer that is also a potential nitrogen fixer, was present only in symbiotic anemones, and its abundance decreased with initial exposure to 34°C, but recovered after 7 days. In aposymbiotic anemones, the added heat stress appeared to result in a large increase of rare bacterial taxa, which included potential pathogens such as Vibrio following bleaching. We also observed several archaea, the first reported for this model, but only in the seawater surrounding aposymbiotic Aiptasia, where abundance increased dramatically following heat stress. We further explored the diazotrophic (nitrogen fixation) potential of diverse bacteria associated with symbiotic and aposymbiotic Aiptasia, under both ambient and heat-stressed conditions, using nifH-PCR and qPCR and the acetylene reduction assay (ARA). In contrast to some stony corals, nifH was barely expressed in both anemone types, and under ambient conditions, diazotrophic activity was not detectable via ARA. Thus, although this research contributes to the growing knowledge of the bacterial community associated with a prominent model used in coral-symbiosis research, our results also suggest using caution when making direct comparisons between Aiptasia and different coral species in microbiome studies.

Nodularia spumigena is a toxic, filamentous cyanobacterium capable of fixing atmospheric N2, which is often dominating cyanobacterial bloom events in the Baltic Sea and other brackish water systems worldwide. Increasing phosphate limitation has been considered as one environmental factor promoting cyanobacterial mass developments. In the present study, we analyzed the response of N. spumigena strain CCY9414 toward strong phosphate limitation. Growth of the strain was diminished under P-deplete conditions; however, filaments contained more polyphosphate under P-deplete compared to P-replete conditions. Using RNA-seq, gene expression was compared in N. spumigena CCY9414 after 7 and 14 days in P-deplete and P-replete conditions, respectively. After 7 days, 112 genes were significantly up-regulated in P-deplete filaments, among them was a high proportion of genes encoding proteins related to P-homeostasis such as transport systems for different P species. Many of these genes became also up-regulated after 14 days compared to 7 days in filaments grown under P-replete conditions, which was consistent with the almost complete consumption of dissolved P in these cultures after 14 days. In addition to genes directly related to P starvation, genes encoding proteins for bioactive compound synthesis, gas vesicles formation, or sugar catabolism were stimulated under P-deplete conditions. Collectively, our data describe an experimentally validated P-stimulon in N. spumigena CCY9414 and provide the indication that severe P limitation could indeed support bloom formation by this filamentous strain. [ABSTRACT FROM AUTHOR]

Cyanobacterial N2‐fixing microorganisms (diazotrophs) play a critical role in nitrogen and carbon cycling in the oceans; hence, accurate measurements of diazotroph abundance are imperative for understanding ocean biogeochemistry. Marine diazotroph abundances are often assessed using qPCR of the nifH gene, a sensitive, taxa‐specific, and time/cost‐efficient method. However, the validity of nifH abundance as a proxy for cell concentration has recently been questioned. Here, we compare nifH gene abundances to cell counts for four diazotroph taxa (Trichodesmium, Crocosphaera, Richelia, and Calothrix) on two cruises to the North Pacific Subtropical Gyre, one of the largest habitats for marine diazotrophs. nifH:cell relationships were strong and significant for Crocosphaera, Richelia, and Calothrix (nifH:cell 1.51–2.58; R2 = 0.89–0.96) but were not significant for Trichodesmium, despite previous studies reporting significant nifH:cell relationships for this organism. Limited available data suggest that empirical nifH:cell can vary among studies but that relationships are usually significantly linear and >1:1. Our study indicates that nifH gene abundance, while not a direct measure of cells, is a useful quantitative proxy for diazotroph abundance. [ABSTRACT FROM AUTHOR]

Background and Aims: Nitrogen (N2) fixation through endophytic diazotrophs within the non-nodulated plants has been considered a novel source of N inputs in terrestrial ecosystems. However, little is known about the composition and diversity of endophytic diazotrophs within the non-nodulated plants, especially when compared with the diazotrophs in nodulated plants. Methods: High-throughput nifH amplification sequencing was conducted to characterize endophytic diazotrophs within different tissues (leaf, stem, and root) of non-nodulated plants (Salix rehderiana Schneid, S. ernestii Schneid, and Populus purdomii Rehd.) and nodulated plants (Astragalus mahoshanicus and Hippophae rhamnoides L.) which dominate in the newly formed N-limited glacier floodplain in the eastern Qinghai-Tibetan Plateau. Results: We found endophytic diazotrophs, primarily Proteobacteria, in all tissue types of non-nodulated plants and leaf and stem of nodulated plants. The dominant genera were Bradyrhizobium, Ideonella, Azotobacter, and Geobacter. In comparison, diazotrophs within the nodule of leguminous Astragalus mahoshanicus and actinorhizal Hippophae rhamnoides were predominantly of genera Rhizobium and Frankia, respectively. Further, the community composition of diazotrophs was structured by plant species (P = 0.001), tissue types (P = 0.001), and nutrient concentrations (P = 0.001). Leaves of non-nodulated plants exhibited higher Chao1 indices and lower Simpson and Shannon indices than their stem and root, but these were not significantly different among different tissue types of nodulated plants. Conclusions: Endophytic diazotrophs might be prevalent in pioneer plants occurring in the N-limited habitat and have a high degree of host specificity; these findings could help to understand the ability of those pioneer plants to acquire N. [ABSTRACT FROM AUTHOR]

Diazotrophic bacteria isolated from the rhizosphere of a wild wheat ancestor, grown from its refuge area in the Fertile Crescent, were found to be efficient Plant Growth-Promoting Rhizobacteria (PGPR), upon interaction with an elite wheat cultivar. In nitrogen-starved plants, they increased the amount of nitrogen in the seed crop (per plant) by about twofold. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar, and to analyze the exo-metabolomes and exo-proteomes. Altered root development was observed, with distinct responses depending on the strain, for instance, with respect to root hair development. A first conclusion from these results is that the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have undergone systematic deep reprogramming during domestication. Exo-metabolome analysis revealed a complex set of secondary metabolites, including nutrient ion chelators, cyclopeptides that could act as phytohormone mimetics, and quorum sensing molecules having inter-kingdom signaling properties. The exo-proteome-comprised strain-specific enzymes, and structural proteins belonging to outer-membrane vesicles, are likely to sequester metabolites in their lumen. Thus, the methodological processes we have developed to collect and analyze bacterial exudates have revealed that PGPRs constitutively exude a highly complex set of metabolites; this is likely to allow numerous mechanisms to simultaneously contribute to plant growth promotion, and thereby to also broaden the spectra of plant genotypes (species and accessions/cultivars) with which beneficial interactions can occur. [ABSTRACT FROM AUTHOR]

Nodularia spumigena is a toxic, filamentous cyanobacterium capable of fixing atmospheric N2, which is often dominating cyanobacterial bloom events in the Baltic Sea and other brackish water systems worldwide. Increasing phosphate limitation has been considered as one environmental factor promoting cyanobacterial mass developments. In the present study, we analyzed the response of N. spumigena strain CCY9414 toward strong phosphate limitation. Growth of the strain was diminished under P-deplete conditions; however, filaments contained more polyphosphate under P-deplete compared to P-replete conditions. Using RNA-seq, gene expression was compared in N. spumigena CCY9414 after 7 and 14 days in P-deplete and P-replete conditions, respectively. After 7 days, 112 genes were significantly up-regulated in P-deplete filaments, among them was a high proportion of genes encoding proteins related to P-homeostasis such as transport systems for different P species. Many of these genes became also up-regulated after 14 days compared to 7 days in filaments grown under P-replete conditions, which was consistent with the almost complete consumption of dissolved P in these cultures after 14 days. In addition to genes directly related to P starvation, genes encoding proteins for bioactive compound synthesis, gas vesicles formation, or sugar catabolism were stimulated under P-deplete conditions. Collectively, our data describe an experimentally validated P-stimulon in N. spumigena CCY9414 and provide the indication that severe P limitation could indeed support bloom formation by this filamentous strain.

Major advances in understanding the diversity, distribution, and activity of marine N2-fixing microorganisms (diazotrophs) have been made in the past decades, however, large gaps in knowledge remain about the environmental controls on growth and mortality rates. In order to measure diazotroph net growth rates and microzooplankton grazing rates on diazotrophs, nutrient perturbation experiments and dilution grazing experiments were conducted using free-floating in situ incubation arrays in the vicinity of Station ALOHA in March 2016. Net growth rates for targeted diazotroph taxa as well as Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes were determined under high (H) and low (L) nitrate:phosphate (NP) ratio conditions at four depths in the photic zone (25, 45, 75, and 100 m) using quantitative PCR and flow cytometry. Changes in the prokaryote community composition in response to HNP and LNP treatments were characterized using 16S rRNA variable region tag sequencing. Microzooplankton grazing rates on diazotrophs were measured using a modified dilution technique at two depths in the photic zone (15 and 125 m). Net growth rates for most of the targeted diazotrophs after 48 h were not stimulated as expected by LNP conditions, rather enhanced growth rates were often measured in HNP treatments. Interestingly, net growth rates of the uncultivated prymnesiophyte symbiont UCYN-A1 were stimulated in HNP treatments at 75 and 100 m, suggesting that N used for growth was acquired through continuing to fix N2 in the presence of nitrate. Net growth rates for UCYN-A1, UCYN-C, Crocosphaera sp. (UCYN-B) and the diatom symbiont Richelia (associated with Rhizosolenia) were uniformly high at 45 m (up to 1.6 ± 0.5 d-1), implying that all were growing optimally at the onset of the experiment at that depth. Differences in microzooplankton grazing rates on UCYN-A1 and UCYN-C in 15 m waters indicate that the grazer assemblage preyed preferentially on UCYN-A1. Deeper in the water column (125 m), both diazotrophs were grazed at substantial rates, suggesting grazing pressure may increase with depth in the photic zone. Constraining in situ diazotroph growth and mortality rates are important steps for improving parameterization for diazotrophs in global ecosystem models.

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.

The present communication deals with the biodiversity and distribution pattern of unbranched, filamentous, heterocystous, diazotrophic, heteropolar and uniseriate blue green algal genus Calothrix Agardh ex Bornet et Flahault from Tripura. In the present paper, we reported total 19 species of the genus Calothrix including one forma and one var. viz. C. aeruginosa, C. breviarticulata, C. clavata, C. clavatoides, C. columbiana, C. conica, C. cylindrica, C. fusca f. durabilis, C. fusca, C. geitleri, C. geitonos, C. gloeocola, C. gracilis, C. javanica, C. linearis, C. marchica v. intermedia, C. parva, C. scopulorum and C. subsimplex from rice fields and sub-aerial habitats of Kumarghat and Kailashahar area of Unakoti district of Tripura, India. All the strains are new addition to the flora of Tripura. [ABSTRACT FROM AUTHOR]

The eastern Indian Ocean (EIO) is characterized by oligotrophic surface water with abundant dust supply, and thus, this environment is considered to provide a favorable niche for cyanobacterial diazotrophs. However, direct measurements have reported low nitrogen fixation with the dominance of heterotrophic diazotrophs, suggesting that the EIO has unique geophysical features and nutrient supplies controlling diazotrophy, in contrast to those in the Atlantic and Pacific Oceans. In this study, we investigated the activity and community of diazotrophs across the EIO from the Bay of Bengal to the west of Australia. Low nitrogen fixation was observed broadly in the EIO (26.1–75.6 μmol N m−2 d−1), except in the west of Australia (82–286 μmol N m−2 d−1). Although the surface nitrate concentration was low in the EIO, the upward nitrate flux was higher because of the shallower nitracline than that in other oligotrophic oceans. This created widespread conditions with low aeFe:N (supply ratio of aerosol iron to nitrogen), resulting in low nitrogen fixation rates. NifH amplicon sequencing demonstrated the dominance of heterotrophic diazotrophs widely in the EIO, with spatial segregation from cyanobacterial diazotrophs (Trichodesmium, UCYN‐A1). In general, cyanobacterial diazotrophs were abundant in the surface waters of the Bay of Bengal and west of Australia with a high aeFe:N supply, whereas heterotrophic diazotrophs were dominant in other broad regions. This finding demonstrates that the shallow nitracline and associated high upward nitrate supply are key factors for the unique diazotrophy in the EIO. Plain Language Summary: Biological nitrogen fixation is a major source of bioavailable nitrogen in the ocean. The eastern Indian Ocean (EIO) has nitrate‐poor surface waters with a replete dust supply from the continents. Thus, it has been considered to provide a niche preferable for cyanobacterial diazotrophs. However, previous direct measurements have shown low nitrogen fixation and dominance of heterotrophic diazotrophs, suggesting the distinct regulation of diazotrophy in the EIO from that in the Atlantic and Pacific Oceans. Here, we examined the activity and community of diazotrophs in the EIO from the Bay of Bengal to the west of Australia. Nitrogen fixation was generally low in the study area, except in the west of Australia. Low nitrogen fixation was related to shallow nitracline and associated low supply ratio of aerosol iron to nitrogen. We also found the spatial segregations of diazotroph communities in response to regional differences in biogeochemical factors such as temperature and nutrient availability. Our results suggest that the high upward nitrate flux due to the shallow nitracline is an important factor for the unique diazotrophy in the Indian Ocean. Key Points: We broadly found low nitrogen fixation rates and dominance of heterotrophic diazotrophs across the eastern Indian OceanConsistently low nitrogen fixation was attributed to the high upward nitrate flux due to the shallow nitraclineCyanobacterial diazotrophs dominate locally with higher activities in regions with elevated aerosol iron supply against nitrate supply [ABSTRACT FROM AUTHOR]

Diatom–diazotroph associations (DDAs) are one of the most important symbiotic dinitrogen (N2) fixing groups in the oligotrophic ocean. Despite their capability to fix N2, ammonium (NH4+) remains a key nitrogen (N) source for DDAs, and the effect of NH4+ on their metabolism remains elusive. Here, we developed a coarse-grained, cellular model of the DDA with NH4+ uptake and quantified how the level of extracellular NH4+ influences metabolism and nutrient exchange within the symbiosis. The model shows that, under a fixed growth rate, an increased NH4+ concentration may lower the required level of N2 fixation and photosynthesis, and decrease carbon (C) and N exchange. A low-NH4+ environment leads to more C and N in nutrient exchange and more fixed N2 to support a higher growth rate. With higher growth rates, nutrient exchange and metabolism increased. Our study shows a strong effect of NH4+ on metabolic processes within DDAs, and thus highlights the importance of in situ measurement of NH4+ concentrations. [ABSTRACT FROM AUTHOR]

Dinitrogen (N 2 ) fixation is a crucial source of bioavailable nitrogen in carbon-dominated cold seep systems. Previous studies have shown that diazotrophy is not necessarily dependent on sulfate-dependent anaerobic oxidation of methane for energy, and diverse catabolism can fuel the high-energy-demanding process in sediments. However, it remains unclear whether diazotroph can obtain energy by sulfur oxidation in sulfur-rich cold seep water column. Here, field investigations and in situ experiments were conducted in Haima cold seep to examine the effects of diverse sources of dissolved organic matter (DOM) on N 2 fixation, specifically containing sulfur, carbon, nitrogen, and phosphorus. We found that active N 2 fixation occurred in the water column above the Haima cold seep, with the Dechloromonas genus dominating the diazotroph community as revealed by nifH gene using high-throughput sequencing. In situ experiments showed an increased rate of N 2 fixation (1.15- to 12.70-fold compared to that in control group) and a greater relative abundance of the Dechloromonas genus following enrichment with sulfur-containing organic matter. Furthermore, metagenomic assembly and binning revealed that Dechloromonas sp. carried genes related to N 2 fixation ( nifDHK ) and sulfur compound oxidation ( fccAB and soxABCXYZ ), implying that the genus potentially serves as a multifunctional mediator for N 2 fixation and sulfur cycling. Our results provide new insights regarding potential coupling mechanism associated with sulfur-driven N 2 fixation in methane- and sulfide-rich environments., Importance: N2 fixation is an important source of biologically available in carbon-dominated cold seep systems as little nitrogen is released by hydrocarbon seepage, thereby promoting biological productivity and the degradation of non-nitrogenous organic matter. Cold seeps are rich in diverse sources of dissolved organic matter (DOM) derived from the sinking of photosynthetic products in euphotic layer and the release of chemosynthesis products on the seafloor. However, it remains unclear whether N2 fixation is coupled to the metabolic processes of DOM, as determined by e.g., carbon, nitrogen, phosphorus, and sulfur content, for energy acquisition in sulfur-rich cold seeps. In this study, diazotroph community structure and its response to DOM compositions were revealed. Moreover, the metagenomics analysis suggested that Dechloromonas genus plays a dominant role in potential coupling N2 fixation and sulfur oxidation. Our study highlighted that sulfur oxidation in deep-sea cold seeps may serve as an energy source to drive N2 fixation., Competing Interests: The authors declare no conflict of interest.

Nitrogen (N)-fixing organisms, also known as diazotrophs, play a crucial role in N-limited ecosystems by controlling the production of bioavailable N. The carbon-dominated cold-seep ecosystems are inherently N-limited, making them hotspots of N fixation. However, the knowledge of diazotrophs in cold-seep ecosystems is limited compared to other marine ecosystems. In this study, we used multi-omics to investigate the diversity and catabolism of diazotrophs in deep-sea cold-seep bottom waters. Our findings showed that the relative abundance of diazotrophs in the bacterial community reached its highest level in the cold-seep bottom waters compared to the cold-seep upper waters and non-seep bottom waters. Remarkably, more than 98% of metatranscriptomic reads aligned on diazotrophs in cold-seep bottom waters belonged to the genus Sagittula , an alphaproteobacterium. Its metagenome-assembled genome, named Seep-BW-D1, contained catalytic genes ( nifHDK ) for nitrogen fixation, and the nifH gene was actively transcribed in situ . Seep-BW-D1 also exhibited chemosynthetic capability to oxidize C1 compounds (methanol, formaldehyde, and formate) and thiosulfate (S 2 O 3 ). In addition, we observed abundant transcripts mapped to genes involved in the transport systems for acetate, spermidine/putrescine, and pectin oligomers, suggesting that Seep-BW-D1 can utilize organics from the intermediates synthesized by methane-oxidizing microorganisms, decaying tissues from cold-seep benthic animals, and refractory pectin derived from upper photosynthetic ecosystems. Overall, our study corroborates that carbon-dominated cold-seep bottom waters select for diazotrophs and reveals the catabolism of a novel chemosynthetic alphaproteobacterial diazotroph in cold-seep bottom waters.2- ). In addition, we observed abundant transcripts mapped to genes involved in the transport systems for acetate, spermidine/putrescine, and pectin oligomers, suggesting that Seep-BW-D1 can utilize organics from the intermediates synthesized by methane-oxidizing microorganisms, decaying tissues from cold-seep benthic animals, and refractory pectin derived from upper photosynthetic ecosystems. Overall, our study corroborates that carbon-dominated cold-seep bottom waters select for diazotrophs and reveals the catabolism of a novel chemosynthetic alphaproteobacterial diazotroph in cold-seep bottom waters., Importance: Bioavailable nitrogen (N) is a crucial element for cellular growth and division, and its production is controlled by diazotrophs. Marine diazotrophs contribute to nearly half of the global fixed N and perform N fixation in various marine ecosystems. While previous studies mainly focused on diazotrophs in the sunlit ocean and oxygen minimum zones, recent research has recognized cold-seep ecosystems as overlooked N-fixing hotspots because the seeping fluids in cold-seep ecosystems introduce abundant bioavailable carbon but little bioavailable N, making most cold seeps inherently N-limited. With thousands of cold-seep ecosystems detected at continental margins worldwide in the past decades, the significant role of cold seeps in marine N biogeochemical cycling is emphasized. However, the diazotrophs in cold-seep bottom waters remain poorly understood. Through multi-omics, this study identified a novel alphaproteobacterial chemoheterotroph belonging to Sagittula as one of the most active diazotrophs residing in cold-seep bottom waters and revealed its catabolism., Competing Interests: The authors declare no conflict of interest.