nifH Gene Sequences Research Articles

Coupling methane oxidation and N2 fixation under methanogenic conditions in contrasting environments

Microbial methane oxidation under widespread suboxic environment is crucial for understanding methane emission. However, the role of aerobic methanotrophs in mediating methane oxidation and nitrogen fixation is less understood in oxygen-limiting environments. In this study, we identified diazotrophic methanotrophs under oxygen-limited conditions (initial O2 of 6–8 μM) in two contrasting habitats (paddy soil and marine sediment) using DNA-based stable isotope probing combined with amplicon sequencing. Consistently, we documented significant 13CH4 oxidation and 15N2 fixation after 740 days of suboxic isotope labeling. Sequencing analysis revealed the predominance of Methylobacter–affiliated aerobic methanotrophs in the 13C-labeled DNA fractions. These Methylobacter-like OTUs accounted for 97.86 % in paddy soil and 99.49 % in marine sediment of the total pmoA gene sequences; while relative abundances for the nifH gene sequences were 91.59 % in paddy soil and 99.49 % in marine sediment. Taken together, our analyses revealed that Methylobacter is responsible for methane oxidation and nitrogen fixation under oxygen limitation in both habitats, demonstrating convergent emergence of this aerobic methanotroph under oxygen deficiency.

Soil diazotrophs sustain nitrogen fixation under high nitrogen enrichment via adjustment of community composition.

This study examined the changes in soil diazotroph community under different loads of simulated N deposition and analyzed its relationship with N fixation rates in in five forests using high-throughput sequencing. The magnitudes of N fixation rates reduced by low N loads were higher than those by high N loads. Low N loads decreased richness and diversity of diazotroph community, whereas diazotroph community structure remained stable under high N loads. Compared with low N loads, high N loads resulted in a less similarity and overlap of nifH gene sequences among the treatments and a larger adjustment of diazotroph community. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ under high N loads did not have negative impacts on diazotroph community structure and N fixation. Based on these findings, it is urgently needed to incorporate the loads of N deposition and the composition of diazotroph community into terrestrial N-cycling models for accurate understanding of N inputs in forest ecosystems.

High biological N fixation potential dominated by heterotrophic diazotrophs in alpine permafrost rivers on the Qinghai‒Tibet Plateau

Biological nitrogen (N) fixation is a pivotal N source in N-deficient ecosystems. The Qinghai‒Tibet Plateau (QTP) region, which is assumed to be N limited and suboxic, is an ideal habitat for diazotrophs. However, the diazotrophic communities and associated N fixation rates in these high-altitude alpine permafrost QTP rivers remain largely unknown. Herein, we examined diazotrophic communities in the sediment and biofilm of QTP rivers via the nitrogenase (nifH) gene sequencing and assessed their N fixing activities via a 15N isotope incubation assay. Strikingly, anaerobic heterotrophic diazotrophs, such as sulfate- and iron-reducing bacteria, had emerged as dominant N fixers. Remarkably, the nifH gene abundance and N fixation rates increased with altitude, and the average nifH gene abundance (2.57 ± 2.60 × 108 copies g−1) and N fixation rate (2.29 ± 3.36 nmol N g−1d−1) surpassed that documented in most aquatic environments (nifH gene abundance: 1.31 × 105 ∼ 2.57 × 108 copies g−1, nitrogen fixation rates: 2.34 × 10−4 ∼ 4.11 nmol N g−1d−1). Such distinctive heterotrophic diazotrophic communities and high N fixation potential in QTP rivers were associated with low-nitrogen, abundant organic carbon and unique C:N:P stoichiometries. Additionally, the significant presence of psychrophilic bacteria within the diazotrophic communities, along with the enhanced stability and complexity of the diazotrophic networks at higher altitudes, clearly demonstrate the adaptability of diazotrophic communities to extreme cold and high-altitude conditions in QTP rivers. We further determined that altitude, coupled with organic carbon and phosphorus, was the predominant driver shaping diazotrophic communities and their N-fixing activities. Overall, our study reveals high N fixation potential in N-deficient QTP rivers, which provides novel insights into nitrogen dynamics in alpine permafrost rivers.

Comparative analysis of nitrogen content and its influence on actinorhizal nodule and rhizospheric microorganism diversity in three Alnus species.

Alnus spp. (alder) are typical nonleguminous nitrogen-fixing trees that have a symbiotic relationship with Frankia. To explore the differences in nitrogen-fixing microorganisms between three alders (A. cremastogyne, A. glutinosa, and A. formosana) with different chromosome ploidies, the community structure and compositional diversity of potential nitrogen-fixing microorganism in root nodules and rhizosphere soil were comparatively analyzed using 16S rRNA and nitrogenase (nifH) gene sequencing. The nitrogen contents in the root nodules and rhizosphere soil were also determined. The results showed that the contents of total nitrogen and nitrate nitrogen in the root nodules of the three alders are significantly higher than those in the rhizosphere soils, while the ammonium nitrogen content show the opposite trend. The family, genus, and species levels showed obviously differences between root nodules and rhizosphere soils, while there were no significant differences at the classification level between the three alders. At the phylum level, the dominant phyla from 16S rRNA and nifH gene data in the root nodules and rhizosphere soil of the three alders are phylum Actinomycetota and phylum Pseudomonadota, respectively. The LEfSe results showed that there are significant differences in the dominant groups in the root nodules and rhizosphere oil of the three alders. The relative abundances of dominant groups also showed obvious differences between the root nodules and rhizosphere soils of three alders. The relative abundances of Frankia and unclassified_Frankia in root nodules are obviously higher than those in rhizosphere soils, and their relative abundances in A. glutinosa root nodules are significantly higher than those in A. cremastogyne and A. formosana at the genus and species levels. The diversity of potential nitrogen-fixing microorganism from 16S rRNA and nifH gene data in the A. glutinosa root nodules and rhizosphere soils are all higher than those in A. cremastogyne and A. formosana. The results of functional prediction also showed that the OTUs for nitrogen fixation, nitrate respiration, and ureolysis in A. glutinosa root nodules are higher than those in the other two alders. Redundancy analysis revealed that the total nitrogen content mostly affects the Frankia community. Overall, there are significant differences in the community composition and structure of potential nitrogen-fixing microorganism in the root nodules and rhizosphere soils between the three alders. A. glutinosa showed a relatively stronger nitrogen fixation capacity than A. formosana and A. cremastogyne. The results help elucidates how the community structure and nitrogen-fixing ability of potential nitrogen-fixing microorganism differ between alder species and serve as a reference for applying Frankia to alder plantations.

Phylogeny and symbiotic effectiveness of indigenous rhizobial microsymbionts of common bean (Phaseolus vulgaris L.) in Malkerns, Eswatini

In most legumes, the rhizobial symbionts exhibit diversity across different environments. Although common bean (Phaseolus vulgaris L.) is one of the important legumes in southern Africa, there is no available information on the genetic diversity and N2-fixing effectiveness of its symbionts in Malkerns, Eswatini. In this study, we assessed the phylogenetic positions of rhizobial microsymbionts of common bean from Malkerns in Eswatini. The isolates obtained showed differences in morpho-physiology and N2-fixing efficiency. A dendrogram constructed from the ERIC-PCR banding patterns, grouped a total of 88 tested isolates into 80 ERIC-PCR types if considered at a 70% similarity cut-off point. Multilocus sequence analysis using 16S rRNA, rpoB, dnaK, gyrB, and glnII and symbiotic (nifH and nodC) gene sequences closely aligned the test isolates to the type strains of Rhizobium muluonense, R. paranaense, R. pusense, R. phaseoli and R. etli. Subjecting the isolates in this study to further description can potentially reveal novel species. Most of the isolates tested were efficient in fixing nitrogen and elicited greater stomatal conductance and photosynthetic rates in the common bean. Relative effectiveness (RE) varied from 18 to 433%, with 75 (85%) out of the 88 tested isolates being more effective than the nitrate fed control plants.

Microevolutionary patterns in ecotypes of the symbiotic cyanobacterium UCYN-A revealed from a Northwest Atlantic coastal time series.

UCYN-A is a globally important nitrogen-fixing symbiotic microbe often found in colder regions and coastal areas where nitrogen fixation has been overlooked. We present a 3-year coastal Northwest Atlantic time series of UCYN-A by integrating oceanographic data with weekly nifH and16S rRNA gene sequencing and quantitative PCR assays for UCYN-A ecotypes. High UCYN-A relative abundances dominated by A1 to A4 ecotypes reoccurred annually in the coastal Northwest Atlantic. Although UCYN-A was detected every summer/fall, the ability to observe separate ecotypes may be highly dependent on sampling time given intense interannual and weekly variability of ecotype-specific occurrences. Additionally, much of UCYN-A's rarer diversity was populated by short-lived neutral mutational variants, therefore providing insight into UCYN-A's microevolutionary patterns. For instance, rare ASVs exhibited community composition restructuring annually, while also sharing a common connection to a dominant ASV within each ecotype. Our study provides additional perspectives for interpreting UCYN-A intraspecific diversity and underscores the need for high-resolution datasets when deciphering spatiotemporal ecologies within UCYN-A.

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

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

Novel symbiovars ingae, lysilomae and lysilomaefficiens in bradyrhizobia from tree-legume nodules

Inga vera and Lysiloma tree legumes form nodules with Bradyrhizobium spp. from the japonicum group that represent novel genomospecies, for which we describe here using genome data, symbiovars lysilomae, lysilomaefficiens and ingae. Genes encoding Type three secretion system (TTSS) that could affect host specificity were found in ingae but not in lysilomae nor in lysilomaefficiens symbiovars and uptake hydrogenase hup genes (that affect nitrogen fixation) were observed in bradyrhizobia from the symbiovars ingae and lysilomaefficiens. nolA gene was found in the symbiovar lysilomaefficiens but not in strains from lysilomae. We discuss that multiple genes may dictate symbiosis specificity. Besides, toxin-antitoxin genes were found in the symbiosis islands in bradyrhizobia from symbiovars ingae and lysilomaefficiens. A limit (95%) to define symbiovars with nifH gene sequences was proposed here.

Native Rhizobia Improve Plant Growth, Fix N2, and Reduce Greenhouse Emissions of Sunnhemp More than Commercial Rhizobia Inoculants in Florida Citrus Orchards.

Sunnhemp (Crotalaria juncea L.) is an important legume cover crop used in tree cropping systems, where there is increased interest by growers to identify rhizobia to maximize soil nitrogen (N) inputs. We aimed to isolate and identify native rhizobia and compare their capabilities with non-native rhizobia from commercial inoculants to fix atmospheric dinitrogen (N2), produce and reduce nitrous oxide (N2O), and improve plant growth. Phylogenetic analyses of sequences of the 16S rRNA and recA, atpD, and glnII genes showed native rhizobial strains belonged to Rhizobium tropici and the non-native strain to Bradyrhizobium japonicum. Plant nodulation tests, sequencing of nodC and nifH genes, and the acetylene-dependent ethylene production assay confirmed the capacity of all strains to nodulate sunnhemp and fix N2. Inoculation with native rhizobial strains resulted in significant increases in root and shoot weight and total C and N contents in the shoots, and showed greater N2-fixation rates and lower emissions of N2O compared to the non-native rhizobium. Our results suggest that native rhizobia improve plant growth, fix N2, and reduce greenhouse emissions of sunnhemp more than commercial rhizobia inoculants in Florida citrus orchards.

Comparison of bacterial diversity, root exudates and soil enzymatic activities in the rhizosphere of AVP1-transgenic and nontransgenic wheat (Triticum aestivum L.).

Soil microbial communities are among the most diverse communities that might be affected due to transgenic crops. Therefore, risk assessment studies on transgenes are essentially required as any adverse effects may depend not only on the specific gene and crop involved but also on soil conditions. The present study deals with the comparison of bacterial populations, root exudates and activities of soil enzymes in nontransgenic and AVP1-transgenic wheat rhizosphere, overexpressing vacuolar H + pyrophosphatase for salinity and drought stress tolerance. Amounts of organic acids and sugars produced as root exudates and activities of dehydrogenase, phosphatase and protease enzymes in soil solution showed no significant differences in AVP1-transgenic and nontransgenic wheat rhizosphere, except for urease and phenol oxidase activities. The higher copy number of nifH gene showed the abundance of nitrogen-fixing bacteria in the rhizosphere of AVP1-transgenic wheat compared with nontransgenic wheat. nifH gene sequence analysis indicated the common diazotrophic genera Azospirillum, Bradyrhizobium, Rhizobium and Pseudomonas in AVP1-transgenic and nontransgenic wheat except for Zoogloea detected only in nontransgenic wheat. Using 454-pyrosequencing of 16S rRNA gene from soil DNA, a total of 156, 282 sequences of 18 phyla were obtained, which represented bacterial (128,006), Archeal (7928) and unclassified (21,568) sequences. Proteobacteria, Crenarchaeota and Firmicutes were the most abundant phyla in the transgenic and nontransgenic wheat rhizosphere. Further comparison of different taxonomic units at the genus level showed similar distribution in transgenic and nontransgenic wheat rhizospheres. We conclude that the AVP1 gene in transgenic wheat has no apparent adverse effects on the soil environment and different bacterial communities. However, the bacterial community depends on several other factors, not only genetic composition of the host plants. The present research supports introduction and cultivation of transgenic plants in agricultural systems without any adverse effects on indigenous bacterial communities and soil ecosystems.

Dark Diazotrophy during the Late Summer in Surface Waters of Chile Bay, West Antarctic Peninsula.

Although crucial for the addition of new nitrogen in marine ecosystems, dinitrogen (N2) fixation remains an understudied process, especially under dark conditions and in polar coastal areas, such as the West Antarctic Peninsula (WAP). New measurements of light and dark N2 fixation rates in parallel with carbon (C) fixation rates, as well as analysis of the genetic marker nifH for diazotrophic organisms, were conducted during the late summer in the coastal waters of Chile Bay, South Shetland Islands, WAP. During six late summers (February 2013 to 2019), Chile Bay was characterized by high NO3− concentrations (~20 µM) and an NH4+ content that remained stable near 0.5 µM. The N:P ratio was approximately 14.1, thus close to that of the Redfield ratio (16:1). The presence of Cluster I and Cluster III nifH gene sequences closely related to Alpha-, Delta- and, to a lesser extent, Gammaproteobacteria, suggests that chemosynthetic and heterotrophic bacteria are primarily responsible for N2 fixation in the bay. Photosynthetic carbon assimilation ranged from 51.18 to 1471 nmol C L−1 d−1, while dark chemosynthesis ranged from 9.24 to 805 nmol C L−1 d−1. N2 fixation rates were higher under dark conditions (up to 45.40 nmol N L−1 d−1) than under light conditions (up to 7.70 nmol N L−1 d−1), possibly contributing more than 37% to new nitrogen-based production (≥2.5 g N m−2 y−1). Of all the environmental factors measured, only PO43- exhibited a significant correlation with C and N2 rates, being negatively correlated (p < 0.05) with dark chemosynthesis and N2 fixation under the light condition, revealing the importance of the N:P ratio for these processes in Chile Bay. This significant contribution of N2 fixation expands the ubiquity and biological potential of these marine chemosynthetic diazotrophs. As such, this process should be considered along with the entire N cycle when further reviewing highly productive Antarctic coastal waters and the diazotrophic potential of the global marine ecosystem.

Rhizobial diversity is associated with inoculation history at a two-continent scale.

A total of 120 Mesorhizobium strains collected from the central dry zone of Myanmar were analyzed in a pot experiment to evaluate nodulation and symbiotic effectiveness (SE%) in chickpea plants. Phylogenetic analyses revealed all strains belonged to the genus Mesorhizobium according to 16–23S rDNA IGS and the majority of chickpea nodulating rhizobia in Myanmar soils were most closely related to M. gobiense, M. muleiense, M. silamurunense, M. tamadayense and M. temperatum. Around two-thirds of the Myanmar strains (68%) were most closely related to Indian strain IC-2058 (CA-181), which is also most closely related to M. gobiense. There were no strains that were closely related to the cognate rhizobial species to nodulate chickpea: M. ciceri and M. mediterraneum. Strains with diverse 16S–23S rDNA IGS shared similar nodC and nifH gene sequences with chickpea symbionts. Detailed sequence analysis of nodC and nifH found that the strains in Myanmar were somewhat divergent from the group including M. ciceri and were more closely related to M. muleiense and IC-2058. A cross-continent analysis between strains isolated in Australia compared with Myanmar found that there was little overlap in species, where Australian soils were dominated with M. ciceri, M. temperatum and M. huakuii. The only co-occurring species found in both Myanmar and Australia were M. tamadayense and M. silumurunense. Continued inoculation with CC1192 may have reduced diversity of chickpea strains in Australian soils. Isolated strains in Australian and Myanmar had similar adaptive traits, which in some cases were also phylogenetically related. The genetic discrepancy between chickpea nodulating strains in Australia and Myanmar is not only due to inoculation history but to adaptation to soil conditions and crop management over a long period, and there has been virtually no loss of symbiotic efficiency over this time in strains isolated from soils in Myanmar.

Influence of environmental factors on the tropical peatlands diazotrophic communities from the Southern Brazilian Atlantic Rain Forest.

The tropical peatlands of southern Brazil are essential for the maintenance of the Atlantic Rain Forest, one of the 25 hotspots of biodiversity in the world. Although diazotrophic micro-organisms are essential for the maintenance of this nitrogen limited ecosystem, so far studies have focused only on micro-organisms involved in the carbon cycle. In this work, peat samples were collected from three tropical peatland regions during dry and rainy seasons and their chemical and microbial characteristics were evaluated. Our results showed that the structure of the diazotrophic communities in the Brazilian tropical peatlands differs in the evaluated seasons. The abundance of the genus Bradyrhizobium showed to be affected by rainfall and peat pH. Despite the shifts of the nitrogen-fixing population in the tropical peatland caused by seasonality it showed to be constantly dominated by α-Proteobacteria followed by Cyanobacteria. In addition, more than 50% of nifH gene sequences have not been classified, indicating the necessity for more studies in tropical peatland, since the reduction of N supply in the peatlands stimulates the recalcitrant organic matter decomposition performed by peatland micro-organisms, influencing the C stock.

Altitudinal niches of symbiotic, associative and free-living diazotrophs driven by soil moisture and temperature in the alpine meadow on the Tibetan Plateau

Legume-associated symbiotic diazotrophs contribute more to nitrogen (N) fixation than non-symbiotic diazotrophs in many terrestrial ecosystems. However, the percentage of legume biomass is low in alpine meadows on the Tibetan Plateau. Therefore, non-symbiotic diazotrophs may play important roles in N fixation in alpine meadow soils. Moreover, Tibetan alpine meadows are fragile and sensitive to global climate change, and the investigating of the key factor driving soil diazotrophic community still entails several challenges. To address these issues, we investigated diazotrophic spatial distribution and diversity along the elevational gradient between 3200 and 4200 m in the alpine meadow using amplicon sequencing of nifH gene. The result clearly showed that soil moisture and temperature were key factors driving soil diazotrophic community structures. Both altitude and soil depth significantly differentiated diazotrophic community composition. Alpha diversity indices of diazotrophic communities showed unimodal distribution along elevation gradient, strongly affected by soil moisture. Altitudinal niches were occupied by different diazotrophs. Soils at lower elevations were dominated by symbiotic diazotrophs and associative diazotrophs related to high biomass of plant hosts, while those at higher elevations were dominated by free-living psychrophiles such as Polaromonas. Furthermore, high moisture stimulated free-living anaerobes at middle elevations, such as Geobacter and Anaeromyxobacter, while suppressed legumes and symbiotic Mezorhizobium. Soil temperature not only directly affected temperature-sensitive diazotrophs, but also indirectly affected them through plants and soil properties such as pH and ammonium content. Our results suggest that climate change may strongly affect biological nitrogen fixation (BNF), and free-living diazotrophs may play important roles in BNF of alpine meadow system on the Tibetan Plateau.

Newly Isolated Paenibacillus monticola sp. nov., a Novel Plant Growth-Promoting Rhizobacteria Strain From High-Altitude Spruce Forests in the Qilian Mountains, China.

Species in the genus Paenibacillus from special habitats have attracted great attention due to their plant growth-promoting traits. A novel plant growth-promoting rhizobacteria (PGPR) species in the genus Paenibacillus was isolated from spruce forest at the height of 3,150 m in the Qilian Mountains, Gansu province, China. The phylogenetic analysis based on 16S rRNA, rpoB, and nifH gene sequences demonstrated that strain LC-T2T was affiliated in the genus Paenibacillus and exhibited the highest sequence similarity with Paenibacillus donghaensis KCTC 13049T (97.4%). Average nucleotide identity (ANIb and ANIm) and digital DNA–DNA hybridization (dDDH) between strain LC-T2T and P. donghaensis KCTC 13049T were 72.6, 83.3, and 21.2%, respectively, indicating their genetic differences at the species level. These differences were further verified by polar lipids profiles, major fatty acid contents, and several distinct physiological characteristics. Meanwhile, the draft genome analysis provided insight into the genetic features to support its plant-associated lifestyle and habitat adaptation. Subsequently, the effects of volatile organic compound (VOC) emitted from strain LC-T2T on the growth of Arabidopsis were evaluated. Application of strain LC-T2T significantly improved root surface area, root projection area, and root fork numbers by 158.3, 158.3, and 241.2%, respectively, compared to control. Also, the effects of LC-T2T on the growth of white clover (Trifolium repens L.) were further assessed by pot experiment. Application of LC-T2T also significantly improved the growth of white clover with root fresh weight increased over three-folds compared to control. Furthermore, the viable bacterial genera of rhizosphere soil were detected in each treatment. The number of genera from LC-T2T-inoculated rhizosphere soil was 1.7-fold higher than that of control, and some isolates were similar to strain LC-T2T, indicating that LC-T2T inoculation was effective in the rhizosphere soil of white clover. Overall, strain LC-T2T should be attributed to a novel PGPR species within the genus Paenibacillus based on phylogenetic relatedness, genotypic features, and phenotypic and inoculation experiment, for which the name Paenibacillus monticola sp. nov. is proposed.

Overlooked and widespread pennate diatom-diazotroph symbioses in the sea

Persistent nitrogen depletion in sunlit open ocean waters provides a favorable ecological niche for nitrogen-fixing (diazotrophic) cyanobacteria, some of which associate symbiotically with eukaryotic algae. All known marine examples of these symbioses have involved either centric diatom or haptophyte hosts. We report here the discovery and characterization of two distinct marine pennate diatom-diazotroph symbioses, which until now had only been observed in freshwater environments. Rhopalodiaceae diatoms Epithemia pelagica sp. nov. and Epithemia catenata sp. nov. were isolated repeatedly from the subtropical North Pacific Ocean, and analysis of sequence libraries reveals a global distribution. These symbioses likely escaped attention because the endosymbionts lack fluorescent photopigments, have nifH gene sequences similar to those of free-living unicellular cyanobacteria, and are lost in nitrogen-replete medium. Marine Rhopalodiaceae-diazotroph symbioses are a previously overlooked but widespread source of bioavailable nitrogen in marine habitats and provide new, easily cultured model organisms for the study of organelle evolution.

Ecological adaptation and phylogenetic analysis of microsymbionts nodulating Polhillia, Wiborgia and Wiborgiella species in the Cape fynbos, South Africa

Polhillia, Wiborgia and Wiborgiella species are shrub legumes endemic to the Cape fynbos of South Africa. They have the ability to fix atmospheric N2 when in symbiosis with soil bacteria called ‘rhizobia’. The aim of this study was to assess the morpho-physiological and phylogenetic characteristics of rhizobia associated with the nodulation of Polhillia, Wiborgia and Wiborgiella species growing in the Cape fynbos. The bacterial isolates from root nodules consisted of a mixture of fast and intermediate growers that differed in colony shape and size. The isolates exhibited tolerance to salinity (0.5–3% NaCl) and pH (pH 5–10) and different antibiotic concentrations, and could produce 0.51 to 51.23 µg mL−1 of indole-3-acetic acid (IAA), as well as solubilize tri-calcium phosphate. The ERIC-PCR results showed high genomic diversity in the rhizobial population and grouped them into two major clusters. Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, nifH and nodC gene sequences revealed distinct and novel evolutionary lineages related to the genus Rhizobium and Mesorhizobium, with some of them being very close to Mesorhizobium australicum. However, the phylogenetic analysis of glnII and nifH genes of some isolates showed incongruency.

Enhancing Symbiotic Nitrogen Fixation and Soybean Growth through Co-Inoculation with Bradyrhizobium and Pseudomonas Isolates

The present study was undertaken to explore acidotolerant rhizobial and non-rhizobial bacteria associated with root nodules of soybean (Glycine max L.). Genotypic and phenotypic characterization regarding nitrogen fixation, nodulation and other potentially plant growth-promotion traits were performed in several isolates. Influences of bacterial inoculation on symbiotic nitrogen fixation and soybean growth were evaluated through flask and pot experiments in a greenhouse. The highest nitrogen-fixing activity was recorded in isolate Bra6, which was closely related to Bradyrhizobium diazoefficiens based on 16S rDNA, nifH, and nodD gene sequences. All the non-rhizobial Pseudomonas isolates possessed multiple plant growth-promoting traits, with various hydrolytic patterns toward plant constituents. In sterile water agar-containing flasks, Bra6 + Pse2 treatment significantly (p &lt; 0.05) increased the number of nodules, fresh weight, and dry weight of both root and shoot. This also led to the increment of most of the nutrients in the soybean plant compared with the uninoculated control or sole inoculation of Bra6. In non-sterile strongly acidic soil-containing pots, co-inoculation with Bra6 and various Pseudomonas isolates showed distinctively positive effects on symbiotic nitrogen fixation and soybean growth. The highest symbiotic nitrogen-fixing activity; root and shoot biomass; as well as N, P, K, Ca, Mg, S, Mn, Cu, and Zn contents of soybean plant were observed in Bra6 + Pse2 treatment. Synergistic symbiosis occurred through co-inoculation with Bradyrhizobium and Pseudomonas isolates, which further enhanced nutrients’ acquisition and growth of soybean in the strongly acidic soils.

Plant-microbe-microbe interactions influence the faba bean nodule colonization by diverse endophytic bacteria.

Legume root nodules harbor rhizobia and other non-nodulating endophytes known as nodule-associated bacteria (NAB) whose role in the legume symbiosis is still unknown. We analysed the genetic diversity of 34 NAB isolates obtained from the root nodules of faba bean grown under various soil conditions in Egypt using 16S rRNA and concatenated sequences of three housekeeping genes. All isolates were identified as members of the family Enterobacteriaceae belonging to the genera Klebsiella, Enterobacter and Raoultella. We identified nine enterobacterial genospecies, most of which have not been previously reported as NAB. All isolated strains harbored nifH gene sequences and most of them possessed plant growth-promoting (PGP) traits. Upon co-inoculation with an N2 fixing rhizobium (Rlv NGB-FR128), two strains (Enterobacter sichanensis NGB-FR97 and Klebsiella variicola NGB-FR116) significantly increased nodulation, growth and N-uptake of faba bean plants over the single treatments or the uninoculated control. The presence of these enterobacteria in nodules was significantly affected by the host plant genotype, symbiotic rhizobium genotype and endophyte genotype, indicating that the nodule colonization process is regulated by plant-microbe-microbe interactions. This study emphasizes the importance of nodule-associated enterobacteria and suggests their potential role in improving the effectiveness of rhizobial inoculants.

Biological Nitrogen Fixation in Sediments of a Cyanobacterial Bloom‐Occurring Bay in One Eutrophic Shallow Lake: Occurrence and Related Environmental Factors

AbstractBiological nitrogen fixation (BNF) is an important source of bioavailable nitrogen in the aquatic environment that supports primary production. However, little is known about its occurrence and potential in eutrophic freshwater habitats. This study investigated the diazotrophic diversity and seasonal variation of nitrogenase activity (NA) in the sediments of a cyanobacterial bloom‐occurring bay of a freshwater lake. Sequencing analysis of the nifH gene revealed that diazotrophs in Clusters I and III constituted the dominant components of the diazotrophic community in Meiliang Bay. Both nifH and 16S rRNA gene sequencing indicated that the microbes in sediments have different community structures during the winter than during other seasons. The concentrations of dissolved sulfide, sulfate, and nitrate in pore water were significantly correlated with NA in sediments; however, the Mantel test indicated that dissolved sulfide had a more significant effect on the structure of the diazotrophic community than did other environmental factors. Laboratory culture experiments under different redox conditions further confirmed the inhibitory effect of dissolved sulfide on BNF in sediments. This study revealed that the BNF in sediments of Meiliang Bay was closely linked to variations in dissolved sulfide concentration, which has broadened our understanding of BNF in freshwater lakes.