Diazotrophic Community Structure Research Articles

Changes in diazotrophic community structure associated with Kuroshio succession in the northern South China Sea

Abstract. Kuroshio intrusion (KI) is a key process that transports water from the western Pacific Ocean to the northern South China Sea (nSCS), where KI-induced surface water mixing often causes variations in microbial assemblages. Yet, how interannual KIs affect the biogeography of diazotrophs and associated environmental factors remains poorly characterized. Here, by quantifying the degree of KIs in 2 consecutive years, coupled with monitoring the diversity and distribution of nitrogenase-encoding nifH phylotypes with quantitative PCR and high-throughput sequencing, we show that changes in the diazotrophic community structure in the nSCS are highly correlated with KI-induced variations in a range of physicochemical parameters. Specifically, the filamentous cyanobacteria in the genus Trichodesmium were more abundant at stations strongly affected by KI and thereby with a deeper mixed layer and higher surface salinity and temperature; the unicellular N2-fixing cyanobacteria in group B (UCYN-B) were more abundant at stations least affected by KI and correlated with nutrient availability, whereas UCYN-C and the γ-proteobacteria were prevalent at stations moderately affected by KI. The neutral community model further demonstrated that dominant diazotrophic subcommunities were significantly affected by environmental factors in 2017 when KI was stronger compared to 2018 when KI retreated. Our analyses provide insightful evidence for the role of KI in shaping the diazotrophic community structure primarily as a stochastic process, implying a potential region-scale redistribution of diazotrophs and nitrogen budget, given that KIs are projected to intensify in a future warming ocean.

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.

Impacts of Managed Vegetation Restoration on Arbuscular Mycorrhizal Fungi and Diazotrophs in Karst Ecosystems.

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.

Microbial species pool-mediated diazotrophic community assembly in crop microbiomes during plant development.

Plant-associated diazotrophs strongly relate to plant nitrogen (N) supply and growth. However, our knowledge of diazotrophic community assembly and microbial N metabolism in plant microbiomes is largely limited. Here we examined the assembly and temporal dynamics of diazotrophic communities across multiple compartments (soils, epiphytic and endophytic niches of root and leaf, and grain) of three cereal crops (maize, wheat, and barley) and identified the potential N-cycling pathways in phylloplane microbiomes. Our results demonstrated that the microbial species pool, influenced by site-specific environmental factors (e.g., edaphic factors), had a stronger effect than host selection (i.e., plant species and developmental stage) in shaping diazotrophic communities across the soil-plant continuum. Crop diazotrophic communities were dominated by a few taxa (~0.7% of diazotrophic phylotypes) which were mainly affiliated with Methylobacterium, Azospirillum, Bradyrhizobium, and Rhizobium. Furthermore, eight dominant taxa belonging to Azospirillum and Methylobacterium were identified as keystone diazotrophic taxa for three crops and were potentially associated with microbial network stability and crop yields. Metagenomic binning recovered 58 metagenome-assembled genomes (MAGs) from the phylloplane, and the majority of them were identified as novel species (37 MAGs) and harbored genes potentially related to multiple N metabolism processes (e.g., nitrate reduction). Notably, for the first time, a high-quality MAG harboring genes involved in the complete denitrification process was recovered in the phylloplane and showed high identity to Pseudomonas mendocina. Overall, these findings significantly expand our understanding of ecological drivers of crop diazotrophs and provide new insights into the potential microbial N metabolism in the phyllosphere.IMPORTANCEPlants harbor diverse nitrogen-fixing microorganisms (i.e., diazotrophic communities) in both belowground and aboveground tissues, which play a vital role in plant nitrogen supply and growth promotion. Understanding the assembly and temporal dynamics of crop diazotrophic communities is a prerequisite for harnessing them to promote plant growth. In this study, we show that the site-specific microbial species pool largely shapes the structure of diazotrophic communities in the leaves and roots of three cereal crops. We further identify keystone diazotrophic taxa in crop microbiomes and characterize potential microbial N metabolism pathways in the phyllosphere, which provides essential information for developing microbiome-based tools in future sustainable agricultural production.

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.

Diazotrophic abundance and community structure associated with three meadow plants on the Qinghai-Tibet Plateau.

Symbiotic diazotrophs form associations with legumes and substantially fix nitrogen into soils. However, grasslands on the Qinghai-Tibet Plateau are dominated by non-legume plants, such as Kobresia tibetica. Herein, we investigated the diazotrophic abundance, composition, and community structure in the soils and roots of three plants, non-legume K. tibetica and Kobresia humilis and the legume Oxytropis ochrocephala, using molecular methods targeting nifH gene. Diazotrophs were abundantly observed in both bulk and rhizosphere soils, as well as in roots of all three plants, but their abundance varied with plant type and soil. In both bulk and rhizosphere soils, K. tibetica showed the highest diazotroph abundance, whereas K. humilis had the lowest. In roots, O. ochrocephala and K. humilis showed the highest and the lowest diazotroph abundance, respectively. The bulk and rhizosphere soils exhibited similar diazotrophic community structure in both O. ochrocephala and K. tibetica, but were substantially distinct from the roots in both plants. Interestingly, the root diazotrophic community structures in legume O. ochrocephala and non-legume K. tibetica were similar. Diazotrophs in bulk and rhizosphere soils were more diverse than those in the roots of three plants. Rhizosphere soils of K. humilis were dominated by Actinobacteria, while rhizosphere soils and roots of K. tibetica were dominated by Verrumicrobia and Proteobacteria. The O. ochrocephala root diazotrophs were dominated by Alphaproteobacteria. These findings indicate that free-living diazotrophs abundantly and diversely occur in grassland soils dominated by non-legume plants, suggesting that these diazotrophs may play important roles in fixing nitrogen into soils on the plateau.

Biological nitrogen fixation, diversity and community structure of diazotrophs in two mosses in 25 temperate forests.

Many moss species are associated with nitrogen (N)-fixing bacteria (diazotrophs) that support the N supply of mosses. Our knowledge relates primarily to pristine ecosystems with low atmospheric N input, but knowledge of biological N fixation (BNF) and diazotrophic communities in mosses in temperate forests with high N deposition is limited. We measured BNF rates using the direct stable isotope method and studied the total and potentially active diazotrophic communities in two abundant mosses, Brachythecium rutabulum and Hypnum cupressiforme, both growing on lying deadwood trunks in 25 temperate forest sites. BNF rates in both mosses were similar to those observed in moss species of pristine ecosystems. H. cupressiforme fixed three times more N2 and exhibited lower diazotrophic richness than B. rutabulum. Frankia was the most prominent diazotroph followed by cyanobacteria Nostoc. Manganese, iron, and molybdenum contents in mosses were positively correlated with BNF and diazotrophic communities. Frankia maintained high BNF rates in H. cupressiforme and B. rutabulum even under high chronic N deposition in Central European forests. Moss N concentration and 15 N abundance indicate a rather minor contribution of BNF to the N nutrition of these mosses.

Biological nitrogen fixation and the role of soil diazotroph niche breadth in representative terrestrial ecosystems

Biological nitrogen fixation (BNF) driven by diazotrophs is a fundamental process underpinning the primary productivity of terrestrial ecosystems and plays a pivotal role in the global nitrogen cycle. However, the relationships between BNF function and soil diazotroph taxa with different niche breadths remain unclear. This study investigated the N2 fixation rate and community structure of diazotrophs in soils collected from representative terrestrial ecosystems in China—agricultural, desert, forest, grassland and wetland—and explored the specific contributions of groups of diazotrophs with different niche breadths to the N2 fixation rate. Among all the ecosystems, the N2 fixation rate, nifH gene abundance and diazotroph diversity were highest in the wetland ecosystems. The N2 fixation rate was significantly (P < 0.05) positively correlated with diazotroph diversity, indicating a relationship between microbial community diversity and ecosystem functionality. Furthermore, the N2 fixation rate and Shannon index were significantly (P < 0.05) positively correlated with the abundances of diazotrophs classified as specialists and opportunists. Structural equation modeling indicated that geographical factors and climatic factors affected the abundance of specialists and opportunists directly or indirectly through soil physicochemical properties, which in turn affected N2 fixation rates. Collectively, these valuable insights into the intrinsic relationship between nitrogen-fixing function and groups of soil diazotroph taxa with different niche breadths advance our understanding of BNF across terrestrial ecosystems.

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.

High site elevation enhanced nitrogen fixation and the stability of diazotrophic community in planted Sonneratia apetala mangrove sediments

Site elevation was decisive in determining effective mangrove restoration, and nitrogen availability was a decisive factor for mangrove growth and development. The biological nitrogen fixation conducted by diazotrophs was responsible for 40–60 % of the nitrogen demand of mangrove ecosystems, and the nitrogen dynamics of mangrove forests were primarily correlated with stand age. Understanding how site elevation affects sediment diazotrophs and their variation across stand age was critical for site selection for mangrove restoration and evaluating the functional rehabilitation and primary productivity of mangrove wetlands. Diazotrophic community and nitrogen fixation rate (NFR) in mangrove sediments with depth were compared among 12-year and 18-year-old S. apetala mangrove forests planted under high (H18) and low site elevations (L18 and L12), respectively. The results indicated the sediment nutrient pools (e.g., TOC, TN, and TS) and NFR in mangrove sediments followed an increasing trend with increased site elevation over time (H18 > L18 > L12), and NFR weakly increased with sediment depth. Moreover, the diazotrophic community structure was both elevation- and age-dependent. The relative abundance of the dominant diazotrophic group Desulfuromonadales (belonged to sulfate-reducing bacteria, SRB) decreased with increasing site elevation, while another SRB group Syntrophobacterales showed an opposite trend. Moreover, network analyses revealed more stable and less complicated networks in sediments with higher site elevation, which followed a similar trend with NFR by H18 > L18 > L12. This study provided new perspectives on the microbe-based assessment of mangrove functional restoration from the viewpoint of both diazotrophic communities and NFR. From this perspective, high site elevation had a higher nutrient pool, NFR, and diazotrophic community stability, which should be recommended for mangrove restoration in the future, and long-term rehabilitation would benefit the ecological functional (nitrogen fixation) restoration.

Soil factors that contribute to the abundance and structure of the diazotrophic community and soybean growth, yield, and quality under biochar amendment

BackgroundA 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.ResultsBiochar 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.ConclusionsSoil 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

Peanut-based Rotation Stabilized Diazotrophic Communities and Increased Subsequent Wheat Yield.

The introduction of legumes into rotations can improve nitrogen use efficiency and crop yield; however, its microbial mechanism involved remains unclear. This study aimed to explore the temporal impact of peanut introduction on microorganisms related to nitrogen metabolism in rotation systems. In this study, the dynamics of diazotrophic communities in two crop seasons and wheat yields of two rotation systems: winter wheat - summer maize (WM) and spring peanut → winter wheat - summer maize (PWM) in the North China Plain were investigated. Our results showed that peanut introduction increased wheat yield and biomass by 11.6% (p < 0.05) and 8.9%, respectively. Lower Chao1 and Shannon indexes of the diazotrophic communities were detected in soils that sampling in June compared with those sampling in September, although no difference was found between WM and PWM. Principal co-ordinates analysis (PCoA) showed that rotation system significantly changed the diazotrophic community structures (PERMANOVA; p < 0.05). Compared with WM, the genera of Azotobacter, Skermanella, Azohydromonas, Rhodomicrobium, Azospirillum, Unclassified_f_Opitutaceae, and Unclassified_f_Rhodospirillaceae were significantly enriched (p < 0.05) in PWM. Furthermore, rotation system and sampling time significantly influenced soil properties, which significantly correlated with the top 15 genera in relative abundance. Partial least squares path modeling (PLS-PM) analysis further showed that the diazotrophic community diversity (alpha- and beta-diversity) and soil properties (pH, SOC and TN) significantly affected wheat yield. In conclusion, legume inclusion has the potential to stabilize diazotrophic community structure at the temporal scales and increase subsequent crop yield.

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.

Negative responses of terrestrial nitrogen fixation to nitrogen addition weaken across increased soil organic carbon levels

The traditional view holds that biological nitrogen (N) fixation is energetically expensive and thus, facultative N fixers reduce N fixation rates while obligate N fixers are excluded by non-N fixers as soil N becomes rich. This view, however, contradicts the phenomenon that N fixation does not decline in many terrestrial ecosystems under N enrichment. To address this paradoxical phenomenon, we conducted a meta-analysis of N fixation and diazotroph (N-fixing microorganism) community structure in response to N addition across terrestrial ecosystems. N addition inhibited N fixation, but the inhibitory effect weakened across increased soil organic carbon (SOC) concentrations. The response ratios of N fixation (including free-living, plant-associated, and symbiotic types) to N addition were lower in the ecosystems with low SOC concentrations (<10 mg/g) than in those with medium or high SOC concentrations (10–20 and > 20 mg/g, respectively). The negative N-addition effects on diazotroph abundance and diversity also weakened across increased SOC levels. Among the climatic and soil factors, SOC was the most important predictor regarding the responses of N fixation and diazotroph community structure to N addition. Overall, our study reveals the role of SOC in affecting the responses of N fixation to N addition, which helps understand the relationships of biological N fixation and N enrichment as well as the mechanisms of terrestrial C and N coupling.

Long-term chemical fertilization results in a loss of temporal dynamics of diazotrophic communities in the wheat rhizosphere

Diazotrophs are potential bacterial biofertilizers with efficacy for plant nutrition, which convert atmospheric N2 into plant available nitrogen. Although they are known to respond strongly to fertilization, little is known about the temporal dynamics of diazotrophic communities throughout plant developmental under different fertilization regimes. In this study, we investigated diazotrophic communities in the wheat rhizosphere at four developmental stages under three long-term fertilization regimes: no fertilizer (Control), chemical NPK fertilizer only (NPK), and NPK fertilizer plus cow manure (NPKM). Fertilization regime had greater effect (explained of 54.9 %) on diazotrophic community structure than developmental stage (explained of 4.8 %). NPK fertilization decreased the diazotrophic diversity and abundance to one-third of the Control, although this was largely recovered by the addition of manure. Meanwhile, Control treatment resulted in significant variation in diazotrophic abundance, diversity, and community structure (P = 0.001) depending on the developmental stage, while the NPK fertilization resulted in the loss of temporal dynamics of the diazotrophic community (P = 0.330), which could be largely recovered by the addition of manure (P = 0.011). Keystone species identified in this study were quite different among the four developmental stages under Control and NPKM treatment but were similar among stages under NPK treatment. These findings suggest that long-term chemical fertilization not only reduces diazotrophic diversity and abundance, but also results in a loss of temporal dynamics of rhizosphere diazotrophic communities.

Enhancement of nitrogen fixation and diazotrophs by long-term polychlorinated biphenyl contamination in paddy soil

Biological nitrogen fixation (BNF) driven by diazotrophs is a major means of increasing available nitrogen (N) in paddy soil, in addition to anthropogenic fertilization. However, the influence of long-term polychlorinated biphenyl (PCB) contamination on the diazotrophic community and nitrogen fixation in paddy soil is poorly understood. In this study, samples were collected from paddy soil subjected to > 30 years of PCB contamination, and the soil diazotrophic community and N2 fixation rate were evaluated by Illumina MiSeq sequencing and acetylene reduction assays, respectively. The results indicated that high PCB contamination increased diazotrophic abundance and the N2 fixation rate, and altered diazotrophic community structure in the paddy soil. The random forest model demonstrated that the β-diversity of the diazotrophic community was the most significant predictor of the N2 fixation rate. Structure equation modeling identified a specialized keystone diazotrophic ecological cluster, predominated by Bradyrhizobium, Desulfomonile, and Cyanobacteria, as the key driver of N2 fixation. Overall, our findings indicated that long-term PCB contamination enhanced the N2 fixation rate by altering diazotrophic community abundance and structure, which may deepen our understanding of the ecological function of diazotrophs in organic-contaminated soil.

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

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

The response of diazotrophs to nutrient amendment in the South China Sea and western North Pacific

Abstract. The availability of iron (Fe) and phosphorus (P) has been shown to be a key factor regulating rates of nitrogen fixation in the western subtropical Pacific. However, the relative importance of Fe and P at finer spatial scales between the northern South China Sea (NSCS) and the western boundary of the North Pacific is poorly constrained. Furthermore, nutrient limitation of specific diazotroph types has not yet been assessed. Here we investigated these unknowns by (i) carrying out measurements of finer-scale spatial variabilities in N2 fixation rates and diazotroph nifH gene abundances throughout these regions and (ii) conducting eight additional Fe and phosphate addition bioassay experiments where both changes in N2 fixation rates and the nifH gene abundances of specific diazotrophs were measured. Overall, nitrogen fixation rates and nifH gene abundances were lower in the NSCS than around the Luzon Strait and the western North Pacific. The nutrient addition bioassay experiments demonstrated that N2 fixation rates in the central NSCS were co-limited by Fe and P, whereas at the western boundary of the North Pacific they were P-limited. Changes in the abundances of nifH in response to nutrient addition varied in how well they correlated with changes in N2 fixation rates, and in six out of eight experiments the largest responses in nifH gene abundances were dominated by either Trichodesmium or UCYN-B (unicellular diazotrophic cyanobacteria group B). In general, nutrient addition had a relatively restricted impact on the composition of the six phylotypes that we surveyed apart from on UCYN-B. This unicellular cyanobacterium group showed increased contribution to the total nifH gene abundance following P addition at sites where N2 fixation rates were P-limited. Our study provides comprehensive evidence of nutrient controls on N2 fixation biogeography in the margin of the western North Pacific. Future research that more accurately constrains nutrient supply rates to this region would be beneficial for resolving what controls diazotroph community structure.

Effects of Chemical Fertilizer Reduction Combined with Straw Application on Diazotrophic Communities in a Double Rice Cropping System

Based on a three-year field experiment, the effects of reduced chemical fertilizer combined with straw application on paddy yield, soil fertility properties, and community structure of diazotrophs in a double-rice cropping field three years after straw application were examined. Three treatments were applied:conventional fertilizer application (CF), chemical fertilizer reduction combined with a low straw application rate (CFLS, 3 t·hm-2), and a high straw application rate (CFHS, 6 t·hm-2). The results showed that CFLS and CFHS did not significantly reduce rice grain yield (P>0.05); significantly neutralized soil acidification; increased soil microbial biomass carbon and nitrogen, dissolved organic carbon, and organic carbon content (P<0.05); and significantly reduced soil redox potential, ammonium nitrogen, and nitrate nitrogen contents (P<0.05). This was more conducive to improve soil nitrogen use efficiency. Compared with those under the CF treatment, the natural nitrogen fixation functional communities of CFLS and CFHS increased the Shannon, PD, and Evenness indexes (P<0.05) due to the improvement of conditions such as the increase in soil carbon storage and the decrease in acidification degree. The relative abundance of microbial communities with nitrogen fixation, carbon fixation, and plant growth promotion functions such as Ferrigenium, Sulfurivermis, Methylomonas, Methylovulum, Ectothiorhodospira, and Nostoc increased significantly (P<0.05). In conclusion, the reduction in chemical fertilizer combined with 3 t·hm-2 and 6 t·hm-2 straw application was an effective measure to improve the community structure of soil diazotrophs and the potential of soil nitrogen fixation.

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

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