Diazotrophic Community Research Articles

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.

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

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

Influence of different microbial inoculants on nitrogen retention and diazotroph community succession during cotton straw composting

Inoculation with microorganisms is an effective method for promoting compost maturity and quality. This study investigated the effects of different microbial inoculants on compost maturity, nitrogen retention, and the diazotroph community during cotton straw composting. Results revealed that inoculation improved organic matter degradation and compost maturity, increasing the total nitrogen masses by 6.6–33.0% and the total nitrogen by 84.6–112.1%. Particularly, the microbial inoculants containing Anoxybacillus rupiensis, Paenibacillus illinoisensis, and Bacillus velezensis (1:1:1, T1 treatment) exhibited the highest total nitrogen masses and total nitrogen content. Furthermore, inoculation enhanced nitrogenase activity and nifH abundance, and markedly influenced the succession of the diazotroph community, increasing their sensitivity to environmental variables during composting. Furthermore, the total nitrogen, C/N ratios, and moisture content significantly affected the diazotroph community. Results from a network analysis indicated that inoculation affected the co-occurrence patterns among the diazotroph species and increased complexity between diazotrophs. In conclusion, this study provided three promising microbial inoculants for high-quality compost production and a reference for promoting nitrogen retention in cotton straw composting.

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

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

On the Role of the Amazon River for N2 Fixation in the Western Tropical Atlantic

AbstractThe high rates of N2 fixation observed in the Western Tropical Atlantic Ocean are powered, at least in parts, by large influx of nutrients from the Amazon River. To disentangle the impact of the Amazon on different factors controlling N2 fixation in the region, we use a high‐resolution regional model (Regional Oceanic Modeling System‐Biogeochemical Elemental Cycling model) that includes two diazotrophic phytoplankton classes (Trichodesmium and Diatom‐Diazotroph‐Assemblages, DDAs). In our simulations, the Amazon enhances marine N2 fixation by 74% (3.8 Tg N yr−1), with most of the enhancement driven by DDAs that represent 90% of the diazotrophic community in plume waters. To determine how the Amazon creates the conditions in which DDAs thrive, we analyze bottom‐up and top‐down controls on phytoplankton along the plume pathway and how the phytoplankton competition is disrupted by the delivery of nutrients by the river. Along the entire plume pathway, DDAs out‐compete Trichodesmium as their higher maximum growth makes them more efficient in building biomass. Nevertheless, DDAs never account for more than 25% of the total phytoplankton biomass as they face the competition of other non‐diazotrophic phytoplankton types in the use of the riverine nutrients. Overall, DDAs thrive in the offshore plume region where their nutrient uptake advantages (N‐fixation, DOP uptake) is concomitant with a relaxed grazing pressure.

Synergistic effects of diazotrophs and arbuscular mycorrhizal fungi on soil biological nitrogen fixation after three decades of fertilization.

Biological nitrogen (N) fixation (BNF) via diazotrophs is an important ecological process for the conversion of atmospheric N to biologically available N. Although soil diazotrophs play a dominant role in BNF and arbuscular mycorrhizal fungi (AMF) serve as helpers to favor BNF, the response of soil BNF and diazotrophic communities to different long-term fertilizations and the role of AMF in diazotrophs-driven BNF are poorly understood. Herein, a 33-year fertilization experiment in a wheat-maize intercropping system was conducted to investigate the changes in soil BNF rates, diazotrophic and AMF communities, and their interactions after long-term representative fertilization (chemical fertilizer, cow manure, wheat straw, and green manure). We found a remarkable increase in soil BNF rates after more than three decades of fertilization compared with nonfertilized soil, and the green manure treatment rendered the highest enhancement. The functionality strengthening was mainly associated with the increase in the absolute abundance of diazotrophs and AMF and the relative abundance of the key ecological cluster of Module #0 (gained from the co-occurrence network of diazotrophic and AMF species) with dominant diazotrophs such as Skermanella and Azospirillum. Furthermore, although the positive correlations between diazotrophs and AMF were reduced under long-term organic fertilization regimes, green manuring could reverse the decline within Module #0, and this had a positive relationship with the BNF rate. This study suggests that long-term fertilization could promote N fixation and select specific groups of N fixers and their helpers in certain areas. Our work provides solid evidence that N fixation and certain groups of diazotrophic and AMF taxa and their interspecies relationship will be largely favored after the fertilized strategy of green manure.

Effects of copper oxide nanoparticles on soil diazotrophic communities in maize rhizosphere

Effects of copper oxide nanoparticles on soil diazotrophic communities in maize rhizosphere

Fertilization regimes affect crop yields through changes of diazotrophic community and gene abundance in soil aggregation

Soil aggregates are extremely vulnerable to agricultural intensification and are important drivers of soil health, microbial diversity, and biogeochemical cycling. Despite its importance, there is a dearth of studies revealing how fertilization regimes influence diazotrophic community behind soil aggregates, as well as the potential consequences for crop yields. To do this, a two-decade fertilization of wheat-maize intercropping field experiment was conducted in Loess Plateau of China semiarid area under three treatments: no fertilizer, chemical and organic fertilizer. Moreover, we categorized soil aggregates as large macroaggregates (>2 mm), medium macroaggregates (1–2 mm), small macroaggregates (0.25–1 mm), microaggregates (< 0.25 mm) and rhizosphere soils aggregates. We found that soil aggregates exerted a much more influence on the nifH gene abundance than fertilization practices. Particularly, nifH gene abundance has been promoted with increasing the size of soil aggregates fraction without blank soil in the organic fertilization while its abundance presented contrast patterns in the chemical fertilization. Bipartite association networks indicated that different soil aggregates shaped niche differentiation of diazotrophic community behind fertilization regimes. Additionally, we found that organic fertilization strengthens the robustness of diazotrophic communities as well as increases the complexity of microbial networks by harboring keystone taxa. Mantel test results suggested that specific soil factors exerted more selective power on diazotrophic community and nifH gene abundance in the chemical fertilization. Furthermore, β-diversity and nifH gene abundance of diazotrophic communities in the soil microaggregates jointly determine the crop yields. Collectively, our findings emphasize the key role of functional community diversity in sustaining soil cycling process and crop yields under long-term fertilization, and facilitate our understanding of the mechanisms underlying diazotrophic community in response to agricultural intensification, which could pave the way to sustainable agriculture through manipulating the functional taxa.

Seasonal Hypoxia Enhances Benthic Nitrogen Fixation and Shapes Specific Diazotrophic Community in the Eutrophic Marine Ranch

Recently, a growing number of studies have confirmed that biological nitrogen fixation is also an important reactive nitrogen source in coastal regions. However, how benthic nitrogen fixation and diazotrophic community in coastal regions respond to seasonal hypoxia remains largely unknown. In this study, we investigated the spatiotemporal pattern of potential nitrogen fixation rate and diazotrophic abundance and community in sediments of a eutrophic marine ranch experiencing summer hypoxia using 15N tracing and high throughput sequencing techniques. The results showed that potential nitrogen fixation rates ranged from 0.013 to 10.199 μmol kg−1 h−1, and were significantly enhanced by summer hypoxia (ANOVA, p &lt; 0.05). However, nifH gene abundance peaked in June. The diazotrophic community was dominated by Geobacteraceae (&gt;60%), followed by Desulfobulbaceae (13.61%). Bottom water oxygen, pH, Chl-a concentration, and sediment NH4+ significantly regulated benthic nitrogen fixation, while the variation of diazotrophic community was explained by sediment TOC, TN, and Fe content (p &lt; 0.05). This study highlighted that hypoxia stimulated benthic nitrogen fixation, which counteracted the nitrogen removal by denitrification and anammox, and could further aggregate eutrophication of the coastal marine ranch. Moreover, the result emphasized the importance of nitrogen fixation in coastal regions for the global N budget.

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.

Associative nitrogen fixation could be common in South African mesic grassland

Non-symbiotic nitrogen-fixing bacterial diazotrophs closely associated with the roots of grasses probably contribute most of the new nitrogen acquired to sustain productive natural grasslands, yet their ecology is poorly understood, especially in southern Africa. We looked for genetic evidence, using qPCR and gel electrophoresis, for the presence of the bacterial nifH gene associated with the roots of four grass species (20 plants each) in a mesic grassland in South Arica, which would indicate the potential for N fixation by diazotrophs. Grasses most tolerant of low N (Aristida junciformis) were predicted to harbour the most diazotrophs, especially compared to those most responsive to fertiliser N (Eragrostis curvula). However, the nifH gene was found in all 80 root samples and did not differ in copy number between species. Sequencing of a representative sample confirmed the identity of the nifH gene. The recently burned half of the grassland had 60% more relative nifH gene copy numbers than the area burned 15 months previously, suggesting that grass growth stimulated by fire could recruit diazotrophs. Given their ubiquity and importance in the N economy of grasslands, research is required to characterise root-associated diazotroph communities, quantify their N fixation rates, and understand their environmental controls.

Revealing the transfer pathways of cyanobacterial-fixed N into the boreal forest through the feather-moss microbiome.

Biological N2 fixation in feather-mosses is one of the largest inputs of new nitrogen (N) to boreal forest ecosystems; however, revealing the fate of newly fixed N within the bryosphere (i.e. bryophytes and their associated organisms) remains uncertain. Herein, we combined 15N tracers, high resolution secondary ion mass-spectrometry (NanoSIMS) and a molecular survey of bacterial, fungal and diazotrophic communities, to determine the origin and transfer pathways of newly fixed N2 within feather-moss (Pleurozium schreberi) and its associated microbiome. NanoSIMS images reveal that newly fixed N2, derived from cyanobacteria, is incorporated into moss tissues and associated bacteria, fungi and micro-algae. These images demonstrate that previous assumptions that newly fixed N2 is sequestered into moss tissue and only released by decomposition are not correct. We provide the first empirical evidence of new pathways for N2 fixed in feather-mosses to enter the boreal forest ecosystem (i.e. through its microbiome) and discuss the implications for wider ecosystem function.

Soil diazotrophic abundance, diversity, and community assembly mechanisms significantly differ between glacier riparian wetlands and their adjacent alpine meadows.

Global warming can trigger dramatic glacier area shrinkage and change the flux of glacial runoff, leading to the expansion and subsequent retreat of riparian wetlands. This elicits the interconversion of riparian wetlands and their adjacent ecosystems (e.g., alpine meadows), probably significantly impacting ecosystem nitrogen input by changing soil diazotrophic communities. However, the soil diazotrophic community differences between glacial riparian wetlands and their adjacent ecosystems remain largely unexplored. Here, soils were collected from riparian wetlands and their adjacent alpine meadows at six locations from glacier foreland to lake mouth along a typical Tibetan glacial river in the Namtso watershed. The abundance and diversity of soil diazotrophs were determined by real-time PCR and amplicon sequencing based on nifH gene. The soil diazotrophic community assembly mechanisms were analyzed via iCAMP, a recently developed null model-based method. The results showed that compared with the riparian wetlands, the abundance and diversity of the diazotrophs in the alpine meadow soils significantly decreased. The soil diazotrophic community profiles also significantly differed between the riparian wetlands and alpine meadows. For example, compared with the alpine meadows, the relative abundance of chemoheterotrophic and sulfate-respiration diazotrophs was significantly higher in the riparian wetland soils. In contrast, the diazotrophs related to ureolysis, photoautotrophy, and denitrification were significantly enriched in the alpine meadow soils. The iCAMP analysis showed that the assembly of soil diazotrophic community was mainly controlled by drift and dispersal limitation. Compared with the riparian wetlands, the assembly of the alpine meadow soil diazotrophic community was more affected by dispersal limitation and homogeneous selection. These findings suggest that the conversion of riparian wetlands and alpine meadows can significantly alter soil diazotrophic community and probably the ecosystem nitrogen input mechanisms, highlighting the enormous effects of climate change on alpine ecosystems.

Nodule-associated diazotrophic community succession is driven by developmental phases combined with microhabitat of Sophora davidii.

Nodule-associated nitrogen-fixing microorganisms (diazotrophs) residing in legume root nodules, and they have the potential to enhance legume survival. However, the succession characteristics and mechanisms of leguminous diazotrophic communities remain largely unexplored. We performed a high-throughput nifH amplicon sequencing with samples of root nodules and soil in the three developmental phases (young nodules, active nodules and senescent nodules) of the Sophora davidii (Franch.) Skeels root nodules, aiming to investigate the dynamics of nodule-endophytic diazotrophs during three developmental phases of root nodules. The results demonstrated the presence of diverse diazotrophic bacteria and successional community shifting dominated by Mesorhizobium and Bradyrhizobium inside the nodule according to the nodule development. The relative abundance decreased for Mesorhizobium, while decreased first and then increased for Bradyrhizobium in nodule development from young to active to senescent. Additionally, strains M. amorphae BT-30 and B. diazoefficiens B-26 were isolated and selected to test the interaction between them in co-cultured conditions. Under co-culture conditions: B. diazoefficiens B-26 significantly inhibited the growth of M. amorphae BT-30. Intriguingly, growth of B. diazoefficiens B-26 was significantly promoted by co'culture with M. amorphae BT-30 and could utilize some carbon and nitrogen sources that M. amorphae BT-30 could not. Additionally, the composition of microbial community varied in root nodules, in rhizosphere and in bulk soil. Collectively, our study highlights that developmental phases of nodules and the host microhabitat were the key driving factors for the succession of nodule-associated diazotrophic community.

Diversity, structure, and distribution of bacterioplankton and diazotroph communities in the Bay of Bengal during the winter monsoon.

The Bay of Bengal (BoB) is conventionally believed to be a low productive, oligotrophic marine ecosystem, where the diazotroph communities presumed to play a vital role in adding "new" nitrogen through the nitrogen fixation process. However, the diazotroph communities in the oceanic region of the BoB are still poorly understood though it represents most of the seawater volume. The present study investigated a detailed account of the bacterioplankton community structure and distribution in the oceanic BoB during the winter monsoon using high throughput sequencing targeting the 16S rRNA and nifH genes. Our study observed diverse groups of bacterioplankton communities in the BoB including both cyanobacterial and non-cyanobacterial phylotypes. Cyanobacteria (Prochlorococcus spp. and Synechococcus spp.) and Proteobacteria (mainly α-, γ-, and δ-Proteobacteria) were the most abundant groups within the bacterial communities, possessing differential vertical distribution patterns. Cyanobacteria were more abundant in the surface waters, whereas Proteobacteria dominated the deeper layers (75 m). However, within the diazotroph communities, Proteobacteria (mainly γ-Proteobacteria) were the most dominant groups than Cyanobacteria. Function prediction based on PICRUSt revealed that nitrogen fixation might more active to add fixed nitrogen in the surface waters, while nitrogen removal pathways (denitrification and anammox) might stronger in deeper layers. Canonical correspondence analysis (CCA) indicated that temperature, salinity, and silicate were major environmental factors driving the distribution of bacterial communities. Additionally, phosphate was also an important factor in regulating the diazotroph communities in the surface water. Overall, this study provided detailed information on bacterial communities and their vital role in the nitrogen cycles in oligotrophic ecosystems.

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.

Long-term N-addition alters the community structure of functionally important N-cycling soil microorganisms across global grasslands

Anthropogenic nitrogen (N) input is known to alter the soil microbiome, but how N enrichment influences the abundance, alpha-diversity and community structure of N-cycling functional microbial communities in grasslands remains poorly understood. Here, we collected soils from plant communities subjected to up to 9 years of annual N-addition (10 g N m−2 per year using urea as a N-source) and from unfertilized plots (control) in 30 grasslands worldwide spanning a large range of climatic and soil conditions. We focused on three key microbial groups responsible for two essential processes of the global N cycle: N2 fixation (soil diazotrophs) and nitrification (AOA: ammonia-oxidizing archaea and AOB: ammonia-oxidizing bacteria). We targeted soil diazotrophs, AOA and AOB using Illumina MiSeq sequencing and measured the abundance (gene copy numbers) using quantitative PCR. N-addition shifted the structure of the diazotrophic communities, although their alpha-diversity and abundance were not affected. AOA and AOB responded differently to N-addition. The abundance and alpha-diversity of AOB increased, and their community structure shifted with N-addition. In contrast, AOA were not affected by N-addition. AOA abundance outnumbered AOB in control plots under conditions of low N availability, whereas N-addition favoured copiotrophic AOB. Overall, N-addition showed a low impact on soil diazotrophs and AOA while effects for AOB communities were considerable. These results reveal that long-term N-addition has important ecological implications for key microbial groups involved in two critical soil N-cycling processes. Increased AOB abundance and community shifts following N-addition may change soil N-cycling, as larger population sizes may promote higher rates of ammonia oxidation and subsequently increase N loss via gaseous and soil N-leaching. These findings bring us a step closer to predicting the responses and feedbacks of microbial-mediated N-cycling processes to long-term anthropogenic N-addition in grasslands.

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

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

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.