A <Emphasis Type="Italic">nifH</Emphasis>-based Oligonucleotide Microarray for Functional Diagnostics of Nitrogen-fixing Microorganisms

Nitrogen fixation is an important process in biogeochemical cycles exclusively carried out by prokaryotes, mostly by an evolutionarily conserved nitrogenase protein complex, of which one of the structural genes (nifH) is highly valuable for phylogenetic and diversity analyses. We developed a nifH-based short oligonucleotide microarray (nifH diagnostic microarray) as a rapid tool to effectively monitor nitrogen-fixing diazotrophic populations in a wide range of environments. Taking account of the overwhelming predominance of environmental nifH fragments from uncultivated microorganisms in public databases, our nifH microarray is mainly based on nifH sequences from as yet unidentified prokaryotes. Standard conditions for microarray performance were determined, and criteria for the design of specific oligonucleotides were defined. A primary set of 56 oligonucleotides was validated with fluorescence-labeled single-stranded nifH targets from five reference strains, 26 environmental clones, and artificial mixtures of reference strains. The nifH microarray was applied to analyze the diversity (based on DNA) and activity (based on mRNA) of diazotrophs in roots of wild rice samples from Namibia. Results demonstrated that only a small subset of diazotrophs being present in the sample were actually fixing nitrogen actively. Our data suggest that the developed nifH microarray is a highly reproducible and semiquantitative method for mapping the variability of diazotrophic diversity, allowing rapid comparisons of the relative abundance and activity of diazotrophic prokaryotes in the environment. A further refined nifH microarray comprising of 194 oligonucleotide probes now covers more than 90% of sequences in our nifH database. Electronic Supplementary Material The following supplementary material is available on-line for this article from     

Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races

Several diazotrophic species of Azoarcus spp. occur as endophytes in the pioneer plant Kallar grass. The purpose of this study was to screen Asian wild rice and cultivated Oryza sativa varieties for natural association with these endophytes. Populations of culturable diazotrophs in surface-sterilized roots were characterized by 16S rDNA sequence analysis, and Azoarcus species were identified by genomic fingerprints. A. indigens and Azoarcus sp. group C were detected only rarely, whereas Azoarcus sp. group D occurred frequently in samples of flooded plants: in 75% of wild rice, 80% of land races of O. sativa from Nepal and 33% of modern cultivars from Nepal and Italy. The putatively endophytic populations of diazotrophs differed with the rice genotype. The diversity of cultured diazotrophs was significantly lower in wild rice species than in modern cultivars. In Oryza officinalis (from Nepal) and O. minuta (from the Philippines), Azoarcus sp. group D were the predominant diazotrophic putative endophytes in roots. In contrast, their number was significantly lower in modern cultivars of O. sativa, whereas numbers and diversity of other diazotrophs, such as Azospirillum spp., Klebsiella sp., Sphingomonas paucimobilis, Burkholderia sp. and Azorhizobium caulinodans, were increased. In land races of O. sativa, the diazotrophic diversity was equally high; however, Azoarcus sp. was found in high apparent numbers. Similar differences in populations were also observed in a culture-independent approach comparing a wild rice (O. officinalis) and a modern-type O. sativa plant: in clone libraries of root-associated nitrogenase (nifH) gene fragments, the diazotrophic diversity was lower in the wild rice species. New lineages of nifH genes were detected, e.g. one deeply branching cluster within the anf (iron) nitrogenases. Our studies demonstrate that the natural host range of Azoarcus spp. extends to rice, wild rice species and old varieties being preferred over modern cultivars.     

Analysis of <Emphasis Type="Italic">nifH</Emphasis> gene diversity in red soil amended with manure in Jiangxi,south China

In order to understand the community structure of diazotrophs in red soil and effects of organic manure Application on the structure, four nifH gene libraries were constructed: the control (CK), low manure (LM), High manure (HM), and high manure adding lime (ML). Totally 150 nifH gene clones were screened and grouped into 21 clusters by RFLP analysis. Existence of dominant patterns was observed in all libraries, which counted for over 96% of clones in library HM and about 56∼72% in other three libraries. The nifH sequences of the dominant patterns in all libraries were most similar to sequences of the cyanobacteria. nifH genes showed high diversity in red soil, dispersing throughout the nifH clades (alpha-, beta-, and gamma-Proteobacteria, Firmicutes, cyanobacteria, Verrucomicrobia, and posited group). Bradyrhizobium and Burkholderia were also important diaxotrophs in low fertility soil samples. Low manure treatment increased the Diversity of nifH genes compared with CK and high manure treatments. Manure and lime treatment led to obvious community succession. Total N to available P ratio, total carbon, and K concentrations were the main factors affecting the diversity of diazotrophs in red soil.     

Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize

Recent studies suggest a high diversity of diazotrophic bacteria in maize. However, none of these works have been based on a sufficient number of samples to provide reasonable quantitative estimates of diazotrophic bacterial diversity. Here we present the use of molecular tools and statistical inference to assess diazotrophic bacterial diversity within rhizosphere soils, roots and stems of field grown maize. DNA was isolated from the latter collected from six maize growing regions within the southern most state in Brazil, Rio Grande do Sul. Using conserved primers, nifH Cluster I gene fragments were amplified from each of the three zones, and the products cloned and sequenced. The majority of the sequences were classified within the Proteobacteria with the α-proteobacteria and β-proteobacteria being the most abundant in the rhizosphere soil and stem samples. The γ-proteobacteria were most abundant in rhizosphere soils, less so in roots, and least in the stem samples. According to three different diversity measures, the rhizosphere soil samples possessed greater diazotrophic bacterial diversity than the roots and stems of the maize plants. Only two genera, Azospirillum and Azotobacter, were found in virtually all samples at an abundance of over 1% of the total nifH sequences obtained. Other genera were largely restricted to soil (Methylocystis, Beijerinckia, Geobacter, Rhodovulum, Methylobacterium, Gluconacetobacter, Methylocella, and Delftia), roots (Dechloromonas), or stems (Methylosinus, Raoultella, and Rhizobium). Three genera, Herbaspirillum, Ideonella, and Klebsiella, appeared to dominate in the interior of the plant but were much rarer in soil.     

Molecular Analysis of Diazotroph Diversity in the Rhizosphere of the Smooth Cordgrass, Spartina alterniflora

N₂ fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria (γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.     

Effects of the Inoculation of <Emphasis Type="Italic">Burkholderia vietnamensis</Emphasis> and Related Endophytic Diazotrophic Bacteria on Grain Yield of Rice

During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some “endophytes” were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using ¹⁵N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.     

Diversity of Nitrogen-Fixing Bacteria Associated with Switchgrass in the Native Tallgrass Prairie of Northern Oklahoma

Switchgrass (Panicum virgatum L.) is a perennial C₄ grass native to North America that is being developed as a feedstock for cellulosic ethanol production. Industrial nitrogen fertilizers enhance switchgrass biomass production but add to production and environmental costs. A potential sustainable alternative source of nitrogen is biological nitrogen fixation. As a step in this direction, we studied the diversity of nitrogen-fixing bacteria (NFB) associated with native switchgrass plants from the tallgrass prairie of northern Oklahoma (United States), using a culture-independent approach. DNA sequences from the nitrogenase structural gene, nifH, revealed over 20 putative diazotrophs from the alpha-, beta-, delta-, and gammaproteobacteria and the firmicutes associated with roots and shoots of switchgrass. Alphaproteobacteria, especially rhizobia, predominated. Sequences derived from nifH RNA indicated expression of this gene in several bacteria of the alpha-, beta-, delta-, and gammaproteobacterial groups associated with roots. Prominent among these were Rhizobium and Methylobacterium species of the alphaproteobacteria, Burkholderia and Azoarcus species of the betaproteobacteria, and Desulfuromonas and Geobacter species of the deltaproteobacteria.     

Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified nitrogenase gene (nifH) fragments is a rapid technique for profiling of diazotrophic microbial communities without the necessity of cultures for study. Here, we examined the impact of N-fertilization, plant genotype and environmental conditions on diazotrophic microbial populations in association with roots of rice (Oryza species) by T-RFLP community profiling and found marked effects on the composition of the microbial community. We found a rapid change of the diazotrophic population structure within 15 days after application of nitrogen fertilizer and a strong effect of environmental conditions and plant genotype. Control experiments revealed that phylogenetically distantly related nifH genes were proportionately amplified, and that signal strength reflected the relative abundance of nifH genes in the sample within a 10-fold range of template concentrations. These results clearly demonstrated that our T-RFLP method was suitable to reflect compositional differences in the diazotrophic community in a semiquantitative manner and that the diazotrophic rhizosphere communities of rice are not static but presumably rather highly dynamic.     

Characterization of Novel Plant Growth Promoting Endophytic Bacterium <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> from Wheat Plant

Nine diazotrophic bacteria were isolated from surface-sterilized roots and culms of wheat variety Malviya-234, which is grown with very low or no inputs of nitrogen fertilizer. Out of the nine bacteria, four showed indole acetic acid (IAA) production, and five were positive for P solubilization. One isolate, WM234C-3, showed appreciable level of nitrogenase activity, IAA production, and P solubilization ability, and was further characterized with a view to exploiting its plant growth promoting activity. Based on 16S rDNA sequence analysis, this isolate was identified as Achromobacter xylosoxidans. Diazotrophic nature of this particular isolate was confirmed by Western blot analysis of dinitrogenase reductase and amplification of nifH. Analysis of the nifH sequence showed close homology with typical diazotrophic bacteria. Endophytic nature and cross-infection ability of WM234C-3 were tested by molecular tagging with gusA fused to a constitutive promoter followed by inoculation onto rice seedlings in axenic conditions. At 21 days after inoculation, the roots showed blue staining, the most intense color being at the emergence of lateral roots and root tips. Microscopic observation confirmed colonization of gus-tagged WM234C-3 in the intercellular spaces of cortical as well as vascular zones of roots. Inoculation of gus-tagged WM234C-3 to rice plants resulted in significant increase in root/shoot length, fresh weight, and chlorophyll a content. Plant growth promoting features coupled with cross-infection ability suggest that this endophytic bacterium may be exploited as agricultural agent for various crops after a thorough and critical pathogenicity test.     

Biogeographic patterns of soil diazotrophic communities across six forests in the North America

Soil diazotrophs play important roles in ecosystem functioning by converting atmospheric N₂ into biologically available ammonium. However, the diversity and distribution of soil diazotrophic communities in different forests and whether they follow biogeographic patterns similar to macroorganisms still remain unclear. By sequencing nifH gene amplicons, we surveyed the diversity, structure and biogeographic patterns of soil diazotrophic communities across six North American forests (126 nested samples). Our results showed that each forest harboured markedly different soil diazotrophic communities and that these communities followed traditional biogeographic patterns similar to plant and animal communities, including the taxa–area relationship (TAR) and latitudinal diversity gradient. Significantly higher community diversity and lower microbial spatial turnover rates (i.e. z‐values) were found for rainforests (~0.06) than temperate forests (~0.1). The gradient pattern of TARs and community diversity was strongly correlated (r² > 0.5) with latitude, annual mean temperature, plant species richness and precipitation, and weakly correlated (r² < 0.25) with pH and soil moisture. This study suggests that even microbial subcommunities (e.g. soil diazotrophs) follow general biogeographic patterns (e.g. TAR, latitudinal diversity gradient), and indicates that the metabolic theory of ecology and habitat heterogeneity may be the major underlying ecological mechanisms shaping the biogeographic patterns of soil diazotrophic communities.     

Flavonoids and Growth Hormones Influence Endophytic Colonization and in Planta Nitrogen Fixation by a Diazotrophic Serratia sp. in Rice

Summary Several endophytic diazotrophs were isolated from cultivars of rice and screened for their diazotrophy by nitrogenase assay and amplification of partial nifH gene. Ability of one of the diazotrophic endophytes, Serratia sp. (isolate EDA2 from cultivar ADT36) to colonize the rice seedlings grown in the presence of flavonoids and growth hormones, under gnotobiotic condition was assessed in cultivar ADT36 using a strain marked with transposon-based egfp and Km ^r . The endophytic colonization was monitored through re-isolation from different parts of rice seedlings in LB+Km plates. Addition of the flavonoids quercetin and diadzein to the growth medium increased the extent of endophytic colonization of the conjugant in rice seedlings by colonizing throughout the plant. Population and in planta nitrogenase activity of Serratia in rice seedlings were significantly increased by addition of flavonoids, quercetin and diadzein, whereas growth hormones, IAA and NAA reduced the efficiency of Serratia. The inoculation of Serratia sp. with flavonoids increased the plant biomass and biochemical constituents of rice seedlings under controlled condition.     

Vertical distribution of diazotrophic bacterial community associated with temperature and oxygen gradients in a subtropical reservoir

Nitrogen-fixing microorganisms play important roles in the structure and function of aquatic ecosystems. However, the diversity and distribution of diazotrophic bacteria along the lake depth continuum are so far poorly understood. In this study, we investigated the dynamic variations of diazotrophs in a subtropical deep reservoir during the stratified period. We applied an in-depth biomolecular approach (DGGE, clone libraries, and quantitative real-time PCR) to explore the nitrogenase (nifH) gene diversity and abundance. The diazotrophic community shifted between the oxic/anoxic interface and the nifH diversity increased with depth. The Cyanobacteria, affiliated to the toxic bloom-forming Cylindrospermopsis raciborskii, were the dominant diazotrophic cluster in the surface waters, whereas diazotrophic Alphaproteobacteria were dominant in the bottom waters. The relationships between microbial and environmental factors clearly demonstrated that the temperature gradient and the oxygen concentration affect the heterogeneity of the diazotrophic community, thereby influencing the entire aquatic nitrogen cycle.     

Abundance and diversity of nitrogen-fixing bacteria in rhizosphere and bulk paddy soil under different duration of organic management

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) approaches were used to assess respectively the molecular diversity and quantity of the nifH gene sequences in rhizosphere and bulk paddy soil under conventional management and different duration of organic management (2, 3, 5, 9 years). The phylogenetic distribution of clones based on nifH gene sequence showed that taxonomic groups were consisted of Alphaproteobacteria (27.6%), Betaproteobacteria (24.1%) and Gammaproteobacteria (48.3%). Members of the order Rhizobiales and Pseudomonadales were prevalent among the dominant diazotrophs. When the quantity of the nifH gene sequences was determined by qPCR, 2.27 × 10⁵ to 1.14 × 10⁶ copies/g of soil were detected. Except for 2 years organically managed soil, nifH gene copy numbers in organic soil, both rhizosphere and bulk, were significantly higher than in CM soil. Moreover, nifH gene copy numbers in the organic rhizosphere soil (3, 5, 9 years) were significantly higher than in bulk soil. The abundance and diversity of nitrogen-fixing bacteria tended to increase with duration of organic management but the highest number of nifH gene copies was observed in the rhizosphere and bulk soil of 5 years organic management. In addition, analysis of variance and canonical correspondence analysis (CCA) showed that C/N, C and N were important factors influencing the abundance and community structure of nitrogen-fixing bacterial.     

Diazotrophic diversity in the rhizosphere of two exotic weed plants, Prosopis juliflora and Parthenium hysterophorus

This study is aimed at assessing culturable diazotrophic bacterial diversity in the rhizosphere of Prosopis juliflora and Parthenium hysterophorus, which grow profusely in nutritionally-poor soils and environmentally-stress conditions so as to identify some novel strains for bioinoculant technology. Diazotrophic isolates from Prosopis and Parthenium rhizosphere were characterized for nitrogenase activity by Acetylene Reduction Assay (ARA) and 16S rRNA gene sequencing. Further, the culture-independent quantitative PCR (qPCR) was performed to compare the abundance of diazotrophs in rhizosphere with bulk soils. The proportion of diazotrophs in total heterotrophs was higher in rhizosphere than bulk soils and 32 putative diazotrophs from rhizosphere of two plants were identified by nifH gene amplification. The ARA activity of the isolates ranged from 40 to 95 nmol ethylene h⁻¹ mg protein⁻¹. The 16S rRNA gene analysis identified the isolates to be members of alpha, beta and gamma Proteobacteria and firmicutes. The qPCR assay also confirmed that abundance of nif gene in rhizosphere of these two plants was 10-fold higher than bulk soil.     

Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply

Summary The population size of diazotrophic bacteria naturally associated with the maize rhizosphere can be affected by biotic and environmental factors. In this work we have evaluated the effect of two genotypes of maize, with and without nitrogen fertilization, on the population dynamics and distribution of diazotrophic bacteria associated with maize plants over different plant ontogenic stages. The study was carried out in a field experiment with and without nitrogen fertilization, using two maize cultivars (Santa Helena 8447 and Santa Rosa 3063) previously selected from 32 maize cultivars for the lowest and highest response to nitrogen fertilization, respectively. Microbiological and molecular approaches were used to characterize the diazotrophic bacterial population structure. Bacterial population was assessed by the most probable number using semi-solid N-free media, and by DNA isolation from soil and plant tissue followed by amplification of nifH gene fragments using nested PCR, and by RFLP analysis using the restriction endonucleases TaqI and HaeIII. The dynamics of the diazotrophic bacterial population were affected by the ontogenic stage of the maize plants, but not by the cultivar type. Roots were the preferred site of colonization, independent of cultivar type and growth stage. During the first stage of maize growth, addition of nitrogen fertilizer negatively affected the diazotrophic bacterial population.     

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

The diazotrophic communities in a rice paddy field were characterized by a molecular polyphasic approach including DNA/RNA-DGGE fingerprinting, real time RT-PCR analysis of nifH gene and the measurement of nitrogen fixation activities. The investigation was performed on a diurnal cycle and comparisons were made between bulk and rhizosphere / root soil as well as between fertilized / unfertilized soils. Real time RT-PCR showed no significant difference in the total quantity of nifH expression under the conditions investigated. The functional diversity and dynamics of the nifH gene expressing diazotroph community investigated using RT-PCR-DGGE revealed high diurnal variations, as well as variation between different soil types. Most of the sequence types recovered from the DGGE gels and clone libraries clustered within nifH Cluster I and III (65 different nifH sequences in total). Sequence types most similar to Azoarcus spp., Metylococcus spp., Rhizobium spp., Methylocystis spp., Desulfovibrio spp., Geobacter spp., Chlorobium spp., were abundant and indicate that these species may be responsible for the observed diurnal variation in the diazotrophic community structure in these rice field samples. Previously described diazotrophic cyanobacterial genera in rice fields, such as Nostoc and Cyanothece, were present in the samples but not detectable in RT-PCR assays.     

Evaluation of the diversity of nitrogen-fixing bacteria in soybean rhizosphere by <Emphasis Type="Italic">nifH</Emphasis> gene analysis

Biological nitrogen fixation plays an important role in the nitrogen balance of agricultural ecosystems and provides an essential part of nitrogen nutrition for plants, even in conditions of intensive fertilization. The main agrobiotechnological method for soybean cultivation (Glycine max (L.) Merril) is an application of microbial preparations based on Bradyrhizobium japonicum. Successful inoculation strongly depends on the interactions between the introduced microorganism and the aboriginal rhizosphere microorganisms. To evaluate the composition of diazotrophic communities, a study of the diversity of the molecular marker for nitrogen fixation, the nifH gene, in the samples of soybean rhizosphere soil was carried out. Experiments were performed in the variants when soybean was cultivated without inoculation and after adding bacterial preparations, as well as in enrichment cultures of diazotrophs. The revealed diazotrophic microorganisms demonstrated low level of similarity to the known microorganisms (74–95% identity by nucleotides), and were identified as species of the phyla Firmicutes and Proteobacteria. In the composition of nitrogen-fixing communities in the rhizosphere soil, the microorganisms of the genera Clostridium, Paenibacillus, and Spirochaeta were shown to prevail.