nifH Gene Sequences Research Articles

Arachis hypogaea L. from Acid Soils of Nanyang (China) Is Frequently Associated with Bradyrhizobium guangdongense and Occasionally with Bradyrhizobium ottawaense or Three Bradyrhizobium Genospecies.

Henan Province is a major area of peanut production in China but the rhizobia nodulating the crop in this region have not been described. A collection of 217 strains of peanut rhizobia was obtained from six field sites across four soil types in Henan Province, North China, by using peanut as a trap host under glasshouse conditions. The 217 strains separated into 8 distinct types on PCR-RFLP analysis of their IGS sequences. Phylogenetic analysis of the 16S rRNA, recA, atpD, and glnII genes of 11 representative strains of the 8 IGS types identified Bradyrhizobium guangdongense, B. ottawaense and three novel Bradyrhizobium genospecies. Bradyrhizobium guangdongense was dominant, accounting for 75.0% of the total isolates across the field sites while B. ottawaense covered 5.1% and the three novel Bradyrhizobium genospecies 4.1 to 8.8% of the total. The symbiosis-related nodA and nifH gene sequences were not congruent with the core genes on phylogenetic analysis and separated into three groups, two of which were similar to sequences of Bradyrhizobium spp. isolated from peanut in south-east China and the third identical to that of B. yuanmingense isolated from Lespedeza cuneata in northern China. A canonical correlation analysis between the distribution of IGS genotypes and soil physicochemical characteristics and climatic factors indicated that the occurrence of IGS types/species was mainly associated with soil pH and available phosphorus.

Depth-dependent influence of biochar application on the abundance and community structure of diazotrophic under sugarcane growth.

Despite progress in understanding diazotrophic distribution in surface soils, few studies have investigated the distribution of diazotrophic bacteria in deeper soil layers. Here, we leveraged high-throughput sequencing (HTS) of nifH genes obtained to assess the influence of biochar amended soil (BC) and control (CK), and soil depths (0–20, 20–40 and 40–60 cm) on diazotrophic abundance and community structures, soil enzyme activities and physio-chemical properties. Multivariate ANOVA analysis revealed that soil depth had profound impact on majority of the soil parameters measured than fertilization. Although soil physio-chemical properties, enzymes activities, diazotrophic genera and enriched operational taxonomic units (OTUs) were significantly influenced across the entire soil profiles, we also observed that BC amended soil significantly increased cane stalk height and weight, nitrate (NO3-), ammonium (NH4+), organic matter (OM), total carbon (TC) and available potassium (AK), and enhanced diazotrophic genera in soil depth 0–20 cm compared to CK treatment. Soil TC, total nitrogen (TN), OM and NH4+ were the major impact factors shifting diazotrophic community structures in soil depth 0–20 cm. Overall, these results were more pronounced in 0–20 cm soil depth in BC than CK treatment.

Nodulation of Shepherdia ×utahensis ‘Torrey’ and the Diversity of Symbiotic Frankia Strains

Shepherdia ×utahensis ‘Torrey’ (hybrid buffaloberry) is an actinorhizal plant that can form symbiotic nodules with the actinobacterial genus Frankia. However, little research has been conducted to investigate the presence of Frankia in their nodules and the effects on plant growth. In this study, plants were grown in a Metro-Mix® 820 substrate and inoculated with soils collected from Mohave County, AZ, or in a low organic-matter substrate inoculated with soils from North Logan, UT. The presence of Frankia was quantified using PolF/PolR primers to amplify their nitrogenase (nifH) gene sequences. In the Metro-Mix 820 substrate, plants irrigated with nitrogen (N)-free Hoagland’s solution at pH 6.5 formed nodules at week 12 after experiment initiation, whereas those receiving the same solution with 2 mm ammonium nitrate (NH4NO3) appeared healthy, but no nodules formed. In the low organic-matter substrate, nodules formed in 5 weeks when plants were irrigated with N-free Hoagland’s solution at pH 7.5. Four 300-bp fragments of query sequences (SU1, SU2, SU3, and SU4) were obtained from nodules. When compared with nifH gene sequences reported in the literature using the Basic Local Alignment Search Tool (BLAST), more than 90% similarity to the nifH of Frankia spp. was obtained. The Frankia strains in the nodules shared nifH sequences similar to those of the same host-specific group of Shepherdia. Furthermore, Frankia strains with similar nifH genes have been reported in nodules of Shepherdia argentea (silver buffaloberry). Additionally, Frankia strains belonging to cluster 3 infective strains consisting of Elaeagnaceae and Rhamnaceae infective Frankia showed high similarity to the query sequences. This research demonstrates that nodulation of S. ×utahensis is inhibited at 2 mm NH4NO3. Apart from N, nodule formation may be associated with the substrate type and pH of the nutrient solution. Based on nifH gene sequence amplification, Frankia strains in the root nodules may have the potential to fix atmospheric nitrogen (N2). These Frankia strains have signature gene sequence characteristics of Elaeagnaceae-infective Frankia, suggesting that S. ×utahensis shares Frankia strains similar to its parents.

Seasonal dynamics of soil microbial diversity and functions along elevations across the treeline

Although microbial diversity patterns along elevations have been extensively studied, little is known about whether the patterns are influenced by seasonality. To test the seasonal and elevational effects on microbial communities and functions, we collected soil samples across a mountain gradient above and below the treeline in three seasons (spring, summer and autumn). Microbial diversity based on the sequencing of 16S rRNA, 18S rRNA and nifH genes was measured, and microbial functions represented by soil basal respiration and microbial biomass were analyzed. As expected, we found significant seasonal and elevational effects on microbial α- and β-diversity and functions, and the effects of elevations were greater than seasonal effects. Elevational patterns of microbial β-diversity and functions were not influenced by seasonality. However, the elevational α-diversity patterns showed by specific groups (bacteria, protist and metazoa) changed among seasons. Further, we identified key soil properties (i.e. moisture, total carbon, total nitrogen and nitrate) which had higher seasonal and elevational variations, mainly contributing to the spatiotemporal variations of microbial diversity and functions. The findings of higher soil nutrients, archaeal and metazoan richness, and microbial functions at the treeline elevation, imply a strong edge effect of treeline on microbial diversity and functions. Together, our study highlights that seasonality influences the elevational patterns of soil microbial α-diversity, rather than that of β-diversity and functions, thus provides new insights into the seasonal and elevational effects on microbial communities and functions.

Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1T, 162S2 and 66S1MBT have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1T (=LMG 29930T=HAMBI 3676T) and 66S1MBT (=LMG 31547T=HAMBI 3720T) as type strains, respectively.

Seasonal and long-term effects of nutrient additions and liming on the nifH gene in cerrado soils under native vegetation

SummaryBiological nitrogen fixation (BNF) represents the main input source of N in tropical savannas. BNF could be particularly important for Brazilian savannas (known as Cerrado) that show a highly conservative N cycle. We evaluated the effects of seasonal precipitation and nutrient additions on the nifH gene abundance in soils from a long-term fertilization experiment in a Cerrado's native area. The experiment consists of five treatments: (1) control, (2) liming, (3) nitrogen (N), (4) nitrogen + phosphorus (NP), and (5) phosphorus (P) additions. The nifH gene sequence was related to Bradyrhizobium members. Seasonal effects on N-fixing potential were observed by a decrease in the nifH relative abundance from rainy to dry season in control, N, and NP treatments. A significant reduction in nifH abundance was found in the liming treatment in both seasons. The findings evidenced the multiple factors controlling the potential N-fixing by free-living diazotrophs in these nutrient-limited and seasonally dry ecosystems.

Changes in the biological N2-fixation rates and diazotrophic community as vegetation recovers on abandoned farmland in a karst region of China

The successful regeneration of karst landscapes that have been severely degraded by intensive agriculture relies on the recovery of biological nitrogen fixation (BNF) by diazotrophic communities in soils after nitrogen (N) derived from synthetic fertilizers dissipates through the soil. It is important to understand the mechanisms that drive BNF because there must be enough bioavailable N in soils to support the recovery of native vegetation communities that are best adapted to erosion-prone karst landscapes. In this study, we quantified the response of the diazotrophic population in topsoil (0–10 cm deep) along a vegetation restoration gradient (sloping cropland > abandoned sloping cropland > secondary forest > primary forest) in the Puding Karst Critical Zone Observatory, Guizhou Province, southwest China from the absolute and specific BNF rates, determined using the acetylene reduction method and qPCR and MiSeq sequencing of nifH genes. The nifH phylogenetic diversities increased as the vegetation increased in maturity and were positively correlated with soil moisture and the N to phosphorus ratio (p < 0.05). The diazotrophic community structure changed with the vegetation recovery time (p < 0.05). Rhizobiales accounted for between 38% and 45% of the total nifH gene sequences and were the most abundant order in the vegetation that had been recovering for the longest time. We found that the soil bulk density was a strong control on the diazotroph community structure and was negatively correlated with Rhizobiales. The absolute and specific BNF rates were the highest in primary forest and abandoned sloping cropland, respectively (p < 0.05). The results from stepwise multiple regression showed that 50% of the variation in the absolute BNF rates was explained by SWC, and 55% of the variation in the nifH specific rates was explained by the available N contents and the diazotrophic community structure. We suggest that the recovery of degraded karst could be accelerated by managing the soil physical structure so that the environment is suitable for diazotrophic communities, which would then improve the BNF efficiency.

Diazotrophic Community Variation Underlies Differences in Nitrogen Fixation Potential in Paddy Soils Across a Climatic Gradient in China.

Biological nitrogen (N2) fixation as a source of new N input into the soil by free-living diazotrophs is important for achieving sustainable rice agriculture. However, the dominant environmental drivers or factors influencing N2 fixation and the functional significance of the diazotroph community structure in paddy soil across a climatic gradient are not yet well understood. Thus, we characterized the diazotroph community and identified the ecological predictors of N2 fixation potential in four different climate zones (mid-temperate, warm-temperate, subtropical, and tropical paddy soils) in eastern China. Comprehensive nifH gene sequencing, functional activity detection, and correlation analysis with environmental factors were estimated. The potential nitrogenase activity (PNA) was highest in warm-temperate regions, where it was 6.2-, 2.9-, and 2.2-fold greater than in the tropical, subtropical, and mid-temperate regions, respectively; nifH gene abundance was significantly higher in warm-temperate and subtropical zones than in the tropical or mid-temperate zones. Diazotroph diversity was significantly higher in the tropical climate zone and significantly lower in the mid-temperate zone. Non-metric multidimensional scaling and canonical correlation analysis indicated that paddy soil diazotroph populations differed significantly among the four climate zones, mainly owing to differences in climate and soil pH. Structural equation models and automatic linear models revealed that climate and nutrients indirectly affected PNA by affecting soil pH and diazotroph community, respectively, while diazotroph community, C/P, and nifH gene abundance directly affected PNA. And C/P ratio, pH, and the diazotroph community structure were the main predictors of PNA in paddy soils. Collectively, the differences in diazotroph community structure have ecological significance, with important implications for the prediction of soil N2-fixing functions under climate change scenarios.

Phylogeographic, toxicological and ecological evidence for the global distribution of Raphidiopsis raciborskii and its northernmost presence in Lake Nero, Central Western Russia

Raphidiopsis raciborskii is a freshwater, potentially toxigenic cyanobacterium, originally described as a tropical species that is spreading to northern regions over several decades. The ability of R. raciborskii to produce cyanotoxins – in particular the alkaloid cylindrospermopsin (CYN), which is toxic to humans and animals – is of serious concern. The first appearance of R. raciborskii in Russia was noted in Lake Nero in the summer of 2010. This is the northernmost (57°N) recorded case of the simultaneous presence of R. raciborskii and detection of CYN. In this study, the data from long-term monitoring of the R. raciborskii population, temperature and light conditions in Lake Nero were explored. CYN and cyr/aoa genes present in environmental samples were examined using HPLC/MS-MS and PCR analysis. A R. raciborskii strain (R104) was isolated and its morphology, toxigenicity and phylogeography were studied. It is supposed that the trigger factor for the strong development of R. raciborskii in Lake Nero in summer 2010 may have been the relatively high water temperature, reaching 29–30 °C. Strain R. raciborskii R104 has straight trichomes and can produce akinetes, making it morphologically similar to European strains. Phylogeographic analysis based on nifH gene and 16S-23S rRNA ITS1 sequences showed that the Russian strain R104 grouped together with R. raciborskii strains isolated from Portugal, France, Germany and Hungary. The Russian strain R104 does not contain cyrA and cyrB genes, meaning that it – like all European strains – cannot produce CYN. Thus, while recent invasion of R. raciborskii into Lake Nero has occurred, morphological, genetic, and toxicological data supported the spreading of this cyanobacterium from other European lakes. Detection of CYN and cyr/aoa genes in environmental samples indicated the cyanobacterium Aphanizomenon gracile as a likely producer of CYN in Lake Nero. The article also discusses data on the global biogeography of R. raciborskii. Genetic similarity between R. raciborskii strains isolated from very remote continents might be related to the ancient origin of the cyanobacterium inhabiting the united continents of Laurasia and Gondwana, rather than comparably recent transoceanic exchange between R. raciborskii populations.

Unveiling of active diazotrophs in a flooded rice soil by combination of NanoSIMS and 15N2-DNA-stable isotope probing

The biological nitrogen fixation in planted and nonplanted paddy soils was quantified using a chamber-based 15N2-labeling technique, and the active diazotrophs of soil were assessed by 15N2-DNA-stable isotope probing (SIP). In addition, the nanometer scale secondary ion mass spectrometry (NanoSIMS) was applied to analysis the 15N-enrichment of soil DNA in SIP fractions. 15N2-labeling experiment showed that BNF was 11.33 ± 1.90 kg N ha−1 in the rice-planted soil and 3.55 ± 1.18 kg N ha−1 in the nonplanted soil after 28-day labeling. The biologically fixed 15N was mainly (> 95%) recovered in the surface layer (0–0.5 cm) in the rice-planted soil. High throughput sequencing of nifH genes extracted from surface soil showed that the presence of rice affected the community composition of diazotrophs. The relative abundance of Nostocales and Stigonematales was significantly higher in rice-planted soil than in nonplanted soil (P < 0.05). After CsCl gradient ultracentrifugation, NanoSIMS images clearly showed that 15N was incorporated into soil DNA in the 15N2-labeling SIP gradient fractions. Analyses of nifH genes in 15N-enriched SIP gradient fractions suggested that Nostocales and Stigonematales were the major contributors to BNF in the rice-soil system. Taken together, these results have highlighted the contributions of cyanobacteria to the BNF in paddy fields and improved our understanding of the close relationship between rice plants and cyanobacteria.

The distribution and relative ecological roles of autotrophic and heterotrophic diazotrophs in the McMurdo Dry Valleys, Antarctica.

ABSTRACTThe McMurdo Dry Valleys (MDV) in Antarctica harbor a diverse assemblage of mat-forming diazotrophic cyanobacteria that play a key role in nitrogen cycling. Prior research showed that heterotrophic diazotrophs also make a substantial contribution to nitrogen fixation in MDV. The goals of this study were to survey autotrophic and heterotrophic diazotrophs across the MDV to investigate factors that regulate the distribution and relative ecological roles of each group. Results indicated that diazotrophs were present only in samples with mats, suggesting a metabolic coupling between autotrophic and heterotrophic diazotrophs. Analysis of 16S rRNA and nifH gene sequences also showed that diazotrophs were significantly correlated to the broader bacterial community, while co-occurrence network analysis revealed potential interspecific interactions. Consistent with previous studies, heterotrophic diazotrophs in MDV were diverse, but largely limited to lakes and their outlet streams, or other environments protected from desiccation. Despite the limited distribution, heterotrophic diazotrophs may make a substantial contribution to the nitrogen budget of MDV due to larger surface area and longer residence times of lakes. This work contributes to our understanding of key drivers of bacterial community structure in polar deserts and informs future efforts to investigate the contribution of nitrogen fixation to MDV ecosystems.

Distinct Intra-lake Heterogeneity of Diazotrophs in a Deep Oligotrophic Mountain Lake.

To date, little is known about the diazotrophs in freshwater ecosystems. In this study, we examined the diversity, abundance, and distribution of the diazotrophic community in the deep oligotrophic Lake Fuxian using high-throughput sequencing and quantitative polymerase chain reaction of nifH genes. Our results showed that the diazotrophs in Lake Fuxian were diverse and were distributed among Proteobacteria, Planctomycetes, Cyanobacteria, Verrucomicrobia, Bacteroidetes, Chloroflexi, and other unclassified environmental sequences. For the first time, it is found that Bacteroidetes and Planctomycetes harbor diazotrophs in freshwater ecosystems. The diazotrophic community compositions were significantly different between the littoral and pelagic zones in the surface layer, and they also changed dramatically along the vertical profile. High diazotrophic abundance and diversity were mostly observed in the surface littoral zone, and overall, a significant relationship between nifH gene richness and abundance was observed. The water turbidity, nitrite, and phosphorus were the most important factors explaining the spatial changes in diversity and abundances of this important functional group. The two most dominant operational taxonomic units belonging to Betaroproteobacteria and Planctomycetes demonstrated opposite distribution patterns in abundance that were driven by non-overlapping environmental factors. This study is by far the first to uncover the high diversity and intra-lake heterogeneity of diazotrophs in a freshwater lake and illuminate the controlling factors. It provides the probability of the co-occurrence of N2 fixation and N-loss in particles.

Symbiotic effectiveness and ecologically adaptive traits of native rhizobial symbionts of Bambara groundnut (Vigna subterranea L. Verdc.) in Africa and their relationship with phylogeny

Bambara groundnut (Vigna subterranea L. Verdc.) is an indigenous, drought-tolerant, underutilized African food legume, with the ability to fix atmospheric N2 in symbiosis with soil bacteria called rhizobia. The aim of this study was to assess the morpho-physiological, symbiotic and phylogenetic characteristics of rhizobia nodulating Bambara groundnut in Ghana, Mali and South Africa. The morpho-physiologically diverse isolates tested were also found to exhibit differences in functional efficiency and phylogenetic positions. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR banding patterns, the isolates were grouped into eight major clusters. The concentrations of Ca, Na and K in soils had a significant (p ≤ 0.01) effect on the distribution of rhizobia. Though many isolates were symbiotically very effective, the effectiveness index varied markedly (p ≤ 0.05) among them. Moreover, the isolates also exhibited tolerance to a wide range of NaCl (0.5–7%), streptomycin (50–500 µg.ml−1), and kanamycin (25–150 µg.ml−1) concentrations. Additionally, these isolates could produce 0.02 to 69.71 µg.ml−1 of indole-3-acetic acid (IAA) in tryptophan-supplemented medium, as well as solubilize tri-calcium phosphate. Phylogenetic analysis of these rhizobial isolates using 16S rRNA, atpD, glnII, gyrB, recA and symbiotic (nifH and nodC) gene sequences revealed distinct and novel evolutionary lineages related to the genus Bradyrhizobium, with some of them being very close to Bradyrhizobium vignae, B. kavangense, B. subterraneum, B. elkanii and B. pachyrhizi.

Microsymbionts of forage peanut under different soil and climate conditions belong to a specific group of Bradyrhizobium strains

The use of mixed pastures of grasses with forage peanut (Arachis pintoi) is growing and offers promising results. This leguminous forage plant has a stoloniferous growth habit and tolerates more intense defoliation and shading. However, there are few studies on its symbiosis with nitrogen (N2) fixing bacteria and no studies on genetic identification of its native microsymbionts. The aim of this study was to evaluate phenotypic and genetic diversity and the symbiotic efficiency of bacterial strains isolated from nodules of forage peanut intercropped with grasses in pasture areas. Root nodules were collected from the Belomonte cultivar in Itabela, Bahia, and BRS Mandobi cultivar in Lavras, Minas Gerais, Brazil. The areas of sample collection had contrasting physical and chemical characteristics. Samples of 10 plants per area and 5 nodules per plant were taken. The strains isolated from the nodules were analyzed for culture characteristics (growth period and change in pH of the culture medium), their 16S rRNA, gyrB and nifH genes were partially sequenced, and authentication and analysis of symbiotic efficiency in forage peanut plants were carried out. Both the authentication and symbiotic efficiency experiments were carried out in a greenhouse under axenic conditions, using A. pintoi cv. BRS Mandobi in a completely randomized design with four replications. Authentication was performed in long neck bottles (500 mL) filled with nutrient solution, and symbiotic efficiency was performed in polypropylene seedling plug pots containing a mixture of sand and vermiculite with a nutrient solution. A total of 74 bacterial strains were obtained from isolation of the forage peanut nodules, 40 from Itabela, and 34 from Lavras. Partial sequencing of the 16S rRNA gene was obtained for 48 strains, 30 from Itabela, and 18 from Lavras. The strains isolated from nodules of forage peanut in pasture areas of Itabela, were identified in the following genera: Bradyrhizobium (21), Rhizobium (5), Burkholderia (2), Kocuria (1), and Paenibacillus (1). The strains coming from soils of Lavras, were identified in the following genera: Bradyrhizobium (13), Rhizobium (2), Burkholderia (1), Mucilaginibacter (1), and Enterobacter (1). In the authentication experiment, 17 strains from Itabela, and 13 strains from Lavras, exhibited nodulation. From these authenticated strains, 18 were identified by 16S rRNA sequencing as belonging to the Bradyrhizobium genus. Of these strains, 11 from the two areas exhibited high symbiotic efficiency in A. pintoi, and their results were better than those of the strains currently approved as inoculants for this crop. The non-nodulating strains are probably nodule endophytes. Sequencing of the gyrB housekeeping gene, performed in 13 strains, confirms phylogenetic groups of potential new species among the strains isolated from A. pintoi, which include strains able to nodulate this species, belonging to a specific phylogenetic group of Bradyrhizobium. Sequences of nifH gene of efficient strains belonged to a group different from that formed by non-nodulating strains.

Multiple factors drive the abundance and diversity of the diazotrophic community in typical farmland soils of China.

Biological nitrogen fixation plays an important role in nitrogen cycling by transferring atmospheric N2 to plant-available N in the soil. However, the diazotrophic activity and distribution in different types of soils remain to be further explored. In this study, 152 upland soils were sampled to examine the diazotrophic abundance, nitrogenase activity, diversity and community composition by quantitative polymerase chain reaction, acetylene reduction assay and the MiSeq sequencing of nifH genes, respectively. The results showed that diazotrophic abundance and nitrogenase activity varied among the three soil types. The diazotrophic community was mainly dominated by Bradyrhizobium, Azospirillum, Myxobacter, Desulfovibrio and Methylobacterium. The symbiotic diazotroph Bradyrhizobium was widely distributed among soils, while the distribution of free-living diazotrophs showed large variation and was greatly affected by multiple factors. Crop type and soil properties directly affected the diazotrophic ɑ-diversity, while soil properties, climatic factors and spatial distance together influenced the diazotrophic community. Network structures were completely different among all three types of soils, with most complex interactions observed in the Red soil. These findings suggest that diazotrophs have various activities and distributions in the three soil types, which played different roles in nitrogen input in agricultural soil in China, being driven by multiple environmental factors.

Heterotrophic Bacteria Dominate the Diazotrophic Community in the Eastern Indian Ocean (EIO) during Pre-Southwest Monsoon

The diazotrophic communities play an important role in sustaining primary productivity through adding new nitrogen to oligotrophic marine ecosystems. Yet, their composition in the oligotrophic Indian Ocean is poorly understood. Here, we report the first observation of phylogenetic diversity and distribution of diazotrophs in the Eastern Indian Ocean (EIO) surface water (to 200m) during the pre-southwest monsoon period. Through high throughput sequencing of nifH genes, we identified diverse groups of diazotrophs in the EIO including both non-cyanobacterial and cyanobacterial phylotypes. Proteobacteria (mainly Alpha-, Beta-, and Gamma-proteobacteria) were the most diverse and abundant groups within all the diazotrophs, which accounted for more than 86.9% of the total sequences. Cyanobacteria were also retrieved, and they were dominated by the filamentous non-heterocystous cyanobacteria Trichodesmium spp. Other cyanobacteria such as unicellular diazotrophic cyanobacteria were detected sporadically. Interestingly, our qPCR analysis demonstrated that the depth-integrated gene abundances of the diazotrophic communities exhibited spatial heterogeneity with Trichodesmium spp. appeared to be more abundant in the Bay of Bengal (p< 0.05), while Sagittula castanea (Alphaproteobacteria) was found to be more dominating in the equatorial region and offshores (p< 0.05). Non-metric multidimensional scaling analysis (NMDS) further confirmed distinct vertical and horizontal spatial variations in the EIO. Canonical correspondence analysis (CCA) indicated that temperature, salinity, and phosphate were the major environmental factors driving the distribution of the diazotroph communities. Overall, our study provides the first insight into the diversity and distribution of the diazotrophic communities in EIO. The findings from this study highlight distinct contributions of both non-cyanobacteria and cyanobacteria to N2 fixation. Moreover, our study reveals information that is critical for understanding spatial heterogeneity and distribution of diazotrophs, and their vital roles in nitrogen and carbon cycling.

A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.).

ObjectiveMaize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield.MethodologyBacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction.ResultsCulturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere.ConclusionThe study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere.

Azospirillum ramasamyi sp. nov., a novel diazotrophic bacterium isolated from fermented bovine products.

A Gram-stain-negative, rod-shaped, aerobic bacterium, designated M2T2B2T, was isolated from fermented bovine products in Suwon, Republic of Korea. The strain displayed growth at 15-45 °C (optimum, 28-30 °C), pH 6.0-10.0 (pH 7.0) and 0-2 % (w/v) NaCl (0 %). Colonies were light pink-coloured, round and convex. The cells were positive for oxidase and weakly positive for catalase. The major fatty acids in whole cells of strain M2T2B2T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), followed by summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), summed feature 2 (C12 : 0 aldehyde/unidentified 10.928/C14 : 0 3-OH/iso-C16 : 1 I), C16 : 0, C18 : 1 2-OH, C16 : 0 3-OH and C17 : 1ω6c. The polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, and three unidentified aminolipids. Ubiquinone 10 was the predominant ubiquinone. The DNA G+C content was 68.0 mol%. The strain could fix atmospheric nitrogen, which was evaluated by the acetylene reduction assay. Further, whole genome sequence analysis revealed the presence of a nif gene cluster. Strain M2T2B2T showed the highest 16S rRNA, rpoD and nifH gene sequence similarity to members of the genus Azospirillum, and showed 97.6 % 16S rRNA gene sequence similarity to Azospirillum oryzae COC8T. The phenotypic, phylogenetic and genomic analyses support the proposal of strain M2T2B2T as being a novel species of the genus Azospirillum, for which the name Azospirillumramasamyi sp. nov. is proposed. The type strain is M2T2B2T (=KACC 14063T=NBRC 106460T).

Burkholderia and Paraburkholderia are Predominant Soybean Rhizobial Genera in Venezuelan Soils in Different Climatic and Topographical Regions.

The climate, topography, fauna, and flora of Venezuela are highly diverse. However, limited information is currently available on the characterization of soybean rhizobia in Venezuela. To clarify the physiological and genetic diversities of soybean rhizobia in Venezuela, soybean root nodules were collected from 11 soil types located in different topographical regions. A total of 395 root nodules were collected and 120 isolates were obtained. All isolates were classified in terms of stress tolerance under different concentrations of NaCl and Al3+. The tolerance levels of isolates to NaCl and Al3+ varied. Based on sampling origins and stress tolerance levels, 44 isolates were selected for further characterization. An inoculation test indicated that all isolates showed the capacity for root nodulation on soybean. Based on multilocus sequence typing (MLST), 20 isolates were classified into the genera Rhizobium and Bradyrhizobium. The remaining 24 isolates were classified into the genus Burkholderia or Paraburkholderia. There is currently no evidence to demonstrate that the genera Burkholderia and Paraburkholderia are the predominant soybean rhizobia in agricultural fields. Of the 24 isolates classified in (Para) Burkholderia, the nodD–nodB intergenic spacer regions of 10 isolates and the nifH gene sequences of 17 isolates were closely related to the genera Rhizobium and Bradyrhizobium, respectively. The root nodulation numbers of five (Para) Burkholderia isolates were higher than those of the 20 α-rhizobia. Furthermore, among the 44 isolates tested, one Paraburkholderia isolate exhibited the highest nitrogen-fixation activity in root nodules.

Marileptolyngbya sina gen. nov., sp. nov. and Salileptolyngbya diazotrophicum gen. nov., sp. nov. (Synechococcales, Cyanobacteria), species of cyanobacteria isolated from a marine ecosystem

We isolated two filamentous, non-heterocystous cyanobacteria, namely SCSIO T-2 from Xincun Bay, Hainan Island and SCSIO 43686 from the northern South China Sea, China. The taxonomic positions of the studied strains were determined by a polyphasic approach that used morphological, molecular, and ecological analyses. The two cyanobacterial strains presented different morphology regarding their cell size and sheaths. Sequencing of the 16S rRNA gene of SCSIO T-2 showed 95% similarity with Leptolyngbya sp. CENA 134 (HQ730083) and SCSIO 43686 displayed 97% similarity with Leptolyngbya sp. L21-BG-2 (KJ206339). In addition, the nifH gene sequence analysis of SCSIO T-2 showed 91% similarity with Lyngbya lagerheimii UTEX 1930 (L15550), while SCSIO 43686 had an 88% similarity with Leptolyngbya minuta LEGE 07181 (KC256775). Both strains’ secondary structures of D1-D1’, Box-B, and V3 regions within the 16S–23S internal transcribed spacer (ITS) region were quite distinct from their closely related cyanobacterial strains. In addition, compared with SCSIO 43686, the SCSIO T-2 strain displayed relatively high light utilization yet low sensitivity to light intensity. Based on these characteristics, the name Marileptolyngbya sina SCSIO T-2 is described, and a new genus named Salileptolyngbya is erected to encompass a monophyletic group. We propose Salileptolyngbya diazotrophicum SCSIO 43686 for the type species of this new genus.