The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-Mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound 'fixation threads' (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-trnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (i) aided the genus-wide retention of nodulation, and (ii) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

Nitrogen Fixation and Microbial Communities Associated with Decomposing Seagrass Leaves in Temperate Coastal Waters.

Seagrass meadows play pivotal roles in coastal biochemical cycles, with nitrogen fixation being a well-established process associated with living seagrass. Here, we tested the hypothesis that nitrogen fixation is also associated with seagrass debris in Danish coastal waters. We conducted a 52-day in situ experiment to investigate nitrogen fixation (proxied by acetylene reduction) and dynamics of the microbial community (16S rRNA gene amplicon sequencing) and the nitrogen fixing community (nifH DNA/RNA amplicon sequencing) associated with decomposing Zostera marina leaves. The leaves harboured distinct microbial communities, including distinct nitrogen fixers, relative to the surrounding seawater and sediment throughout the experiment. Nitrogen fixation rates were measurable on most days, but highest on days 3 (dark, 334.8 nmol N g-1 dw h-1) and 15 (light, 194.6 nmol N g-1 dw h-1). Nitrogen fixation rates were not correlated with the concentration of inorganic nutrients in the surrounding seawater or with carbon:nitrogen ratios in the leaves. The composition of nitrogen fixers shifted from cyanobacterial Sphaerospermopsis to heterotrophic genera like Desulfopila over the decomposition period. On the days with highest fixation, nifH RNA gene transcripts were mainly accounted for by cyanobacteria, in particular by Sphaerospermopsis and an unknown taxon (order Nostocales), alongside Proteobacteria. Our study shows that seagrass debris in temperate coastal waters harbours substantial nitrogen fixation carried out by cyanobacteria and heterotrophic bacteria that are distinct relative to the surrounding seawater and sediments. This suggests that seagrass debris constitutes a selective environment where degradation is affected by the import of nitrogen via nitrogen fixation.

Endophytic diazotrophic communities from rice roots are diverse and weakly associated with soil diazotrophic community composition and soil properties.

AIM: Bacteria that promote plant growth, such as diazotrophs, are valuable tools for achieving a more sustainable production of important non-legume crops like rice. Different strategies have been used to discover new bacteria capable of promoting plant growth. This work evaluated the contribution of soil diazotrophs to the endophytic communities established in the roots of rice seedlings cultivated on seven representative soils from Uruguay. METHODS AND RESULTS: The soils were classified into two groups according to the C and clay content. qPCR, terminal restriction fragment length polymorphism (T-RFLP), and 454-pyrosequencing of the nifH gene were used for analyzing diazotrophs in soil and plantlets' roots grown from seeds of the same genotype for 25 days under controlled conditions. A similar nifH abundance was found among the seven soils, roots, or leaves. The distribution of diazotrophs was more uneven in roots than in soils, with dominance indices significantly higher than in soils (nifH T-RFLP). Dominant soils' diazotrophs were mainly affiliated to Alphaproteobacteria and Planctomycetota. Conversely, Alpha, Beta, Gammaproteobacteria, and Bacillota were predominant in different roots, though undetectable in soils. Almost no nifH sequences were shared between soils and roots. CONCLUSIONS: Root endophytic diazotrophs comprised a broader taxonomic range of microorganisms than diazotrophs found in soils from which the plantlets were grown and showed strong colonization patterns.

Bacteria undergo significant shifts while archaea maintain stability in Pocillopora damicornis under sustained heat stress.

Global warming reportedly poses a critical risk to coral reef ecosystems. Bacteria and archaea are crucial components of the coral holobiont. The response of archaea associated with warming is less well understood than that of the bacterial community in corals. Also, there have been few studies on the dynamics of the microbial community in the coral holobiont under long-term heat stress. In order to track the dynamic alternations in the microbial communities within the heat-stressed coral holobiont, three-week heat-stress monitoring was carried out on the coral Pocillopora damicornis. The findings demonstrate that the corals were stressed at 32 °C, and showed a gradual decrease in Symbiodiniaceae density with increasing duration of heat stress. The archaeal community in the coral holobiont remained relatively unaltered by the increasing temperature, whereas the bacterial community was considerably altered. Sustained heat stress exacerbated the dissimilarities among parallel samples of the bacterial community, confirming the Anna Karenina Principle in animal microbiomes. Heat stress leads to more complex and unstable microbial networks, characterized by an increased average degree and decreased modularity, respectively. With the extension of heat stress duration, the relative abundances of the gene (nifH) and genus (Tistlia) associated with nitrogen fixation increased in coral samples, as well as the potential pathogenic bacteria (Flavobacteriales) and opportunistic bacteria (Bacteroides). Hence, our findings suggest that coral hosts might recruit nitrogen-fixing bacteria during the initial stages of suffering heat stress. An environment that is conducive to the colonization and development of opportunistic and pathogenic bacteria when the coral host becomes more susceptible as heat stress duration increases.

Occurrence, identification and characterization of diazotrophic bacteria from aerial roots of Rhynchostylis retusa (L.) Blume for plant growth-promoting activity.

In this study, the diversity of diazotrophic bacteria of orchid Rhynchostylis retusa (L.) Blume and its potential application in plant growth promotion were evaluated. About 183 nitrogen-fixing bacteria were isolated to screen various plant growth-promoting traits viz. phosphate solubilization,IAA, siderophore, HCN, biofilm and ammonia production. Based on 16S rRNA gene sequencing analysis Achromobacter, Arthrobacter, Acinetobacter, Bacillus, Brevibacterium, Curtobacterium, Erwinia, Kosakonia, Lysinibacillus, Klebseilla, Microbacterium, Mixta, Pantoea, Pseudomonas and Stenotrophomonas isolates were selected and showed positive results for PGP traits. Overall, genus Pantoea, Brevibacterium, Achromobacter, Arthrobacter, Klebsiella, Mixta, Bacillus, and Pseudomonas had the most pronounced PGP characteristics and acetylene reduction among the screened isolates. BOX PCR fingerprinting analysis showed variation in polymorphic banding patterns among diazotrophic strains. PCR amplification of nifH gene and the presence of 37 kDa nitrogenase reductase enzyme band in western blot indicated presence of nitrogenase activity. Our study showed that orchid R. retusa diazotroph interaction helps orchid plant to fix nitrogen, essential nutrients, and control pathogen entry. To the best of our knowledge, this is the first report on characterization of diazotrophic bacterial community from aerial roots of R. retusa.

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.

Nitrogen fixation by diverse diazotrophic communities can support population growth of arboreal ants.

BACKGROUND: Symbiotic ant-plant associations, in which ants live on plants, feed on plant-provided food, and protect host trees against threats, are ubiquitous across the tropics, with the Azteca-Cecropia associations being amongst the most widespread interactions in the Neotropics. Upon colonization of Cecropia's hollow internodes, Azteca queens form small patches with plant parenchyma, which are then used as waste piles when the colony grows. Patches-found in many ant-plant mutualisms-are present throughout the colony life cycle and may supplement larval food. Despite their initial nitrogen (N)-poor substrate, patches in Cecropia accommodate fungi, nematodes, and bacteria. In this study, we investigated the atmospheric N2 fixation as an N source in patches of early and established ant colonies. RESULTS: Via 15N2 tracer assays, N2 fixation was frequently detected in all investigated patch types formed by three Azteca ant species. Quantified fixation rates were similar in early and established ant colonies and higher than in various tropical habitats. Based on amplicon sequencing, the identified microbial functional guild-the diazotrophs-harboring and transcribing the dinitrogenase reductase (nifH) gene was highly diverse and heterogeneous across Azteca colonies. The community composition differed between early and established ant colonies and partly between the ant species. CONCLUSIONS: Our data show that N2 fixation can result in reasonable amounts of N in ant colonies, which might not only enable bacterial, fungal, and nematode growth in the patch ecosystems but according to our calculations can even support the growth of ant populations. The diverse and heterogeneous diazotrophic community implies a functional redundancy, which could provide the ant-plant-patch system with a higher resilience towards changing environmental conditions. Hence, we propose that N2 fixation represents a previously unknown potential to overcome N limitations in arboreal ant colonies.

Lupin, a Unique Legume That Is Nodulated by Multiple Microsymbionts: The Role of Horizontal Gene Transfer.

Lupin is a high-protein legume crop that grows in a wide range of edaphoclimatic conditions where other crops are not viable. Its unique seed nutrient profile can promote health benefits, and it has been proposed as a phytoremediation plant. Most rhizobia nodulating Lupinus species belong to the genus Bradyrhizobium, comprising strains that are phylogenetically related to B. cytisi, B. hipponenese, B. rifense, B. iriomotense/B. stylosanthis, B. diazoefficiens, B. japonicum, B. canariense/B. lupini, and B. retamae/B. valentinum. Lupins are also nodulated by fast-growing bacteria within the genera Microvirga, Ochrobactrum, Devosia, Phyllobacterium, Agrobacterium, Rhizobium, and Neorhizobium. Phylogenetic analyses of the nod and nif genes, involved in microbial colonization and symbiotic nitrogen fixation, respectively, suggest that fast-growing lupin-nodulating bacteria have acquired their symbiotic genes from rhizobial genera other than Bradyrhizobium. Horizontal transfer represents a key mechanism allowing lupin to form symbioses with bacteria that were previously considered as non-symbiotic or unable to nodulate lupin, which might favor lupin's adaptation to specific habitats. The characterization of yet-unstudied Lupinus species, including microsymbiont whole genome analyses, will most likely expand and modify the current lupin microsymbiont taxonomy, and provide additional knowledge that might help to further increase lupin's adaptability to marginal soils and climates.

Long-term warming effects on the microbiome and nifH gene abundance of a common moss species in sub-Arctic tundra.

Bacterial communities form the basis of biogeochemical processes and determine plant growth and health. Mosses harbour diverse bacterial communities that are involved in nitrogen fixation and carbon cycling. Global climate change is causing changes in aboveground plant biomass and shifting species composition in the Arctic, but little is known about the response of moss microbiomes in these environments. Here, we studied the total and potentially active bacterial communities associated with Racomitrium lanuginosum in response to a 20-yr in situ warming in an Icelandic heathland. We evaluated the effect of warming and warming-induced shrub expansion on the moss bacterial community composition and diversity, and nifH gene abundance. Warming changed both the total and the potentially active bacterial community structure, while litter abundance only affected the total bacterial community structure. The abundance of nifH genes was negatively affected by litter abundance. We also found shifts in the potentially nitrogen-fixing community, with Nostoc decreasing and noncyanobacterial diazotrophs increasing in relative abundance. Our data suggest that the moss microbial community and potentially nitrogen fixing taxa will be sensitive to future warming, partly via changes in litter and shrub abundance.

Empirical relationship between nifH gene abundance and diazotroph cell concentration in the North Pacific Subtropical Gyre.

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

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.

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.

Unraveling Nitrogen Fixing Potential of Endophytic Diazotrophs of Different Saccharum Species for Sustainable Sugarcane Growth.

Sugarcane (Saccharum officinarum L.) is one of the world's highly significant commercial crops. The amounts of synthetic nitrogen (N2) fertilizer required to grow the sugarcane plant at its initial growth stages are higher, which increases the production costs and adverse environmental consequences globally. To combat this issue, sustainable environmental and economic concerns among researchers are necessary. The endophytic diazotrophs can offer significant amounts of nitrogen to crops through the biological nitrogen fixation mediated nif gene. The nifH gene is the most extensively utilized molecular marker in nature for studying N2 fixing microbiomes. The present research intended to determine the existence of novel endophytic diazotrophs through culturable and unculturable bacterial communities (EDBCs). The EDBCs of different tissues (root, stem, and leaf) of five sugarcane cultivars (Saccharum officinarum L. cv. Badila, S. barberi Jesw.cv Pansahi, S. robustum, S. spontaneum, and S. sinense Roxb.cv Uba) were isolated and molecularly characterized to evaluate N2 fixation ability. The diversity of EDBCs was observed based on nifH gene Illumina MiSeq sequencing and a culturable approach. In this study, 319766 operational taxonomic units (OTUs) were identified from 15 samples. The minimum number of OTUs was recorded in leaf tissues of S. robustum and maximum reads in root tissues of S. spontaneum. These data were assessed to ascertain the structure, diversity, abundance, and relationship between the microbial community. A total of 40 bacterial families with 58 genera were detected in different sugarcane species. Bacterial communities exhibited substantially different alpha and beta diversity. In total, 16 out of 20 genera showed potent N2-fixation in sugarcane and other crops. According to principal component analysis (PCA) and hierarchical clustering (Bray-Curtis dis) evaluation of OTUs, bacterial microbiomes associated with root tissues differed significantly from stem and leaf tissues of sugarcane. Significant differences often were observed in EDBCs among the sugarcane tissues. We tracked and validated the plethora of individual phylum strains and assessed their nitrogenase activity with a culture-dependent technique. The current work illustrated the significant and novel results of many uncharted endophytic microbial communities in different tissues of sugarcane species, which provides an experimental system to evaluate the biological nitrogen fixation (BNF) mechanism in sugarcane. The novel endophytic microbial communities with N2-fixation ability play a remarkable and promising role in sustainable agriculture production.

Diversity of soil nitrogen-fixing bacteria in the rhizosphere and non-rhizophere soils of Ebinur Lake Wetland.

Ebinur Lake Wetland is an understudied desert wetland ecosystem, particularly regarding nitrogen cycling. This study aimed to ascertain the diversity and richness of nitrogen-fixing bacterial communities in the Ebinur Lake Wetland. The diversity of the nitrogen-fixing bacteria community of nifH genes from the rhizosphere and non-rhizosphere soils of four plants in different seasons were examined using Illumina HiSeq PE250 high-throughput sequencing technology. The correlation between soil environmental factors and diversity and richness of nitrogen-fixing bacteria was studied using the redundancy analysis (RDA). The results showed that the diversity of nitrogen-fixing bacteria in the rhizosphere soil of the constructive plants was higher than that in the non-rhizosphere soil; also, the diversity in July was higher than that in October and April. Geobacter, Pseudomonas and Bradyrhizobium were the dominant common bacteria in different samples of Ebinur Lake Wetland. The RDA showed that the total nitrogen, available potassium and available phosphoruswere significantly correlated with the diversity and richness of nitrogen-fixing bacteria. The diversity and community structure of nitrogen-fixing bacteria in soil samples also changed over time. The community structures of nitrogen-fixing bacteria in the rhizosphere and non-rhizosphere soils of the four plants were not the same during the same period. The correlation between soil environmental factors and the community structure and abundance of nitrogen-fixing bacteria can provide data basis and theoretical support for the degradation and restoration of Ebinur Lake Wetland.

Community Composition and Co-Occurrence Patterns of Diazotrophs along a Soil Profile in Paddy Fields of Three Soil Types in China.

Diazotrophs play a key role in biological nitrogen (N2) fixation. However, we know little about the distribution of the diazotrophic community along the soil profile in paddy fields. Here, we used Illumina MiSeq sequencing, targeting the nitrogenase reductase (nifH) gene, to investigate changes with depth (0-100 cm) in the diazotrophic community in paddy soils of three regions (Changshu, Hailun, and Yingtan) in China. The results indicated that most diazotrophs belonged to the phylum Proteobacteria, accounting for 78.05% of the total number of sequences. The diazotrophic diversity was generally highest in the 10-20 cm layer, and then significantly decreased with soil depth. Principal coordinate analysis and PERMANOVA indicated that the diazotrophic community structure was significantly affected by region and soil depth. There were obvious differences in the composition of the diazotrophic community between the topsoil (0-40 cm) and the subsoil (40-100 cm). Anaeromyxobacter, Sideroxydans, Methylomonas, Nostoc, Methanocella, and Methanosaeta were enriched in the topsoil, while Geobacter, Azoarcus, Bradyrhizobium, and Dechloromonas were concentrated in the subsoil. Furthermore, co-occurrence network analysis showed that the diazotrophic network in the topsoil was more complex than that in the subsoil. Distance-based redundancy analysis indicated that soil total C and N content and pH were the main factors influencing the vertical variation in the diazotrophic community. These results highlighted that depth has a great impact on the diazotrophic diversity, community composition, and co-occurrence patterns in paddy soil.

Depicting Temporal, Functional, and Phylogenetic Patterns in Estuarine Diazotrophic Communities from Environmental DNA and RNA.

Seasonally nitrogen-limited and phosphorus-replete temperate coastal waters generally host dense and diverse diazotrophic communities. Despite numerous studies in marine systems, little is known about diazotrophs and their functioning in oligohaline estuarine environments. Here we applied a combination of nifH transcript and metagenomic shotgun sequencing approaches to investigate temporal shifts in taxonomic composition and nifH activity of size-fractionated diazotrophic communities in a shallow and mostly freshwater coastal lagoon. Patterns in active nifH phylotypes exhibited a clear seasonal succession, which reflected their different tolerances to temperature change and nitrogen (N) availability. Thus, in spring, heterotrophic diazotrophs (Proteobacteria) dominated the nifH phylotypes, while increasing water temperature and depletion of inorganic N fostered heterocystous Cyanobacteria in summer. Metagenomic data demonstrated four main N-cycling pathways and three of them with a clear seasonal pattern: denitrification (spring) → N2 fixation (summer) → assimilative NO3- reduction (fall), with NH4+ uptake into cells occurring across all seasons. Although a substantial denitrification signal was observed in spring, it could have originated from the re-suspended benthic rather than planktonic community. Our results contribute to a better understanding of the realized genetic potential of pelagic N2 fixation and its seasonal dynamics in oligohaline estuarine ecosystems, which are natural coastal biogeochemical reactors.

Diversity and abundance of culturable nitrogen-fixing bacteria in the phyllosphere of maize.

AIMS: The present study aimed at gaining an insight into the abundance and genetic diversity of culturable N-fixing epiphyte bacteria on the phyllosphere of maize in arid and semi-arid regions of Iran. METHODS AND RESULTS: Leaf samples of the maize variety, 'single cross 704' (Zea mays L.) were collected from different locations in Iran. The community of culturable N-fixing epiphyte bacteria present was examined by 16S rRNA sequencing, BOXAIR-polymerase chain reaction (PCR) and restricted fragment length polymorphisms analysis of 16S rRNA gene (16S-RFLP). Approximately, 31·82% of the 242 isolates were identified as N-fixers by cultivation of bacteria in Rennie medium and detection of their nifH gene. The N-fixers were affiliated with four bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. 16S rRNA sequencing detected 16 genera and 24 different species in the identified phyla. The most dominant genus was Bacillus and the species identified were B. pumilus, B. amyloliquefaciens, B. subtilis, B. paralicheniformis, B. licheniformis, B. niabensis and B. megaterium. In total, 22 RFLP groups were present among the isolates originally identified as N-fixing bacteria. BOXAIR-PCR showed that there was a low similarity level among the N-fixing bacteria isolates, and genetic differentiation of individual strains was relatively great. CONCLUSIONS: Our findings suggest that nitrogen-fixing epiphyte bacteria on the phyllosphere of maize may provide significant nitrogen input into arid and semi-arid ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: This research implies that phyllosphere epiphyte diazotrophs have much to offer in sustainable agriculture and can be an alternative to chemical N-fertilizers for providing nitrogen to crops arid and semi-arid regions.

Transcriptional analysis of nitrogen fixation in Paenibacillus durus during growth in nitrogen-enriched medium.

Paenibacillus durus strain ATCC 35681T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognizable by SigE, a sigma factor normally involved in sporulation.

Effect of different short-term tillage management on nitrogen-fixing bacteria community in a double-cropping paddy field of southern China.

Soil nitrogen (N)-fixing bacteria community plays an important role in the N cycling process in soil, but there is still limited information about how the soil microbes that drive this process to respond to combined application of tillage and crop residue management under the double-cropping rice (Oryza sativa L.) paddy field in southern of China. Therefore, the effects of 6-years short-term tillage treatment on soil N-fixing bacteria community under the double-cropping rice paddy field in southern China were studied by using the polymerase chain reaction-denaturing gradient gel electrophoresis method. The field experiment included four tillage treatments: conventional tillage with crop residue incorporation (CT), rotary tillage with crop residue incorporation (RT), no-tillage with crop residue retention (NT), rotary tillage with crop residue removed as control (RTO). The results showed that the diversity index and richness index of cbbLR and nifH genes with CT, RT, and NT treatments were increased, compared with RTO treatment. Compared with RTO treatment, the abundance of cbbLR gene with CT, RT, and NT treatments were increased by 6.54, 4.73, and 2.78 times, respectively. Meanwhile, the abundance of nifH gene with CT, RT, and NT treatments were 5.32, 3.71, and 2.45 times higher than that of RTO treatment. The results also indicated that soil autotrophic Azotobacter and nitrogenase activity with CT and RT treatments were significantly higher (p < .05) than that of RTO treatment. There was an obvious difference in characteristic of soil N-fixing bacteria community between the application of crop residue and without crop residue input treatments. In summary, the results indicated that the abundance of N-fixing bacteria community in the double-cropping rice paddy field increased with conventional tillage and rotary tillage practice.

Abundance and diversity of diazotrophs in the surface sediments of Kongsfjorden, an Arctic fjord.

Diazotrophy in the Arctic environment is poorly understood compared to tropical and subtropical regions. Hence in this study, we report the abundance and diversity of diazotrophs in Arctic fjord sediments and elucidate the role of environmental factors on the distribution of diazotrophs. The study was conducted during the boreal summer in the Kongsfjorden, an Arctic fjord situated in the western coast of Spitsbergen. The abundance of nifH gene was measured through quantitative real-time PCR and the diversity of diazotrophs was assessed by nifH targeted clone library and next generation sequence analysis. Results revealed that the abundance of nifH gene in the surface sediments ranged from 2.3 × 106 to 3.7 × 107 copies g- 1. The δ-proteobacterial diazotrophs (71% of total sequence) were the dominant class observed in this study. Major genera retrieved from the sequence analysis were Desulfovibrionaceae (25% of total sequence), Desulfuromonadaceae (18% of total sequence) and Desulfobacteriaceae (10% of total sequence); these are important diazotrophic iron and sulfur-reducing bacterial clade in the Kongsfjorden sediments. The abundance of nifH gene showed a significant positive correlation TOC/TN ratio (r2 = 0.96, p ≤ 0.05) and total organic carbon (p ≤ 0.05) content in the fjord sediments. The higher TOC/TN ratio (4.24-14.5) indicated low nitrogen content organic matter in the fjord sediments through glacier runoff, which enhances the abundance and diversity of nitrogen fixing microorganisms.