Arsenic Contamination in Agricultural Soil Reduces Metabolic Activity of Total and Free-Living Nitrogen-Fixing Bacteria as Revealed by Real-Time qPCR

Laboratory microcosms were used to assess the impact of arsenic (As) contamination on agricultural soil bacterial activity focusing free-living nitrogen fixers for three months. Periodically collected microcosm samples were analyzed by RT–qPCR following extraction of total RNA and cDNA preparation for assessing the metabolically active bacterial population. RT–qPCR data showed the gradual increase of 16S rRNA and nifH gene expression, and relative activity of diazotrophs in the enriched soil bacterial consortia under short time As exposure up to 20 ppm and 10 ppm, respectively. A similar trend of these variables was also noticed but up to 1 ppm As when incubating the bulk soil for the same duration. Reduced bacterial activity was noticed at higher concentration of As although in short time exposure. Extending the As exposure time, the bacterial activities in both enriched consortia and bulk soil were decreased. Although, the relative activity of diazotrophs in enriched consortia was increased in presence of 10 ppm As, the same was decreased in bulk soil when exposed to >1 ppm As for long time indicating susceptibility of nitrogen fixer to As contamination in soil. PCA of the data obtained also indicated a negative correlation between As concentrations and diazotrophic activity.     

Enumeration, isolation and identification of diazotrophs from Korean wetland rice varieties grown with long-term application of N and compost and their short-term inoculation effect on rice plants

AIM: This study has been aimed (i) to isolate and identify diazotrophs from Korean rice varieties; (ii) to examine the long-term effect of N and compost on the population dynamics of diazotrophs and (iii) to realize the shot-term inoculation effect of these diazotrophs on rice seedlings. METHODS AND RESULTS: Diazotrophic and heterotrophic bacterial numbers were enumerated by most probable number method and the isolates were identified based on morphological, physiological, biochemical and 16s rDNA sequence analysis. Long-term application of fertilizer N with compost enhanced both these numbers in rice plants and its environment. Bacteria were high in numbers when malate and azelaic acids were used as carbon source, but less when sucrose was used as a carbon substrate. The combined application promoted the association of diazotrophic bacteria like Azospirillum spp., Herbaspirillum spp., Burkholderia spp., Gluconacetobacter diazotrophicus and Pseudomonas spp. in wetland rice plants. Detection of nifD genes from different diazotrophic isolates indicated their nitrogen fixing ability. Inoculation of a representative isolate from each group onto rice seedlings of the variety IR 36 grown in test tubes indicated the positive effect of these diazotrophs on the growth of rice seedlings though the percentage of N present in the plants did not differ much. CONCLUSIONS: Application of compost with fertilizer N promoted the diazotrophic and heterotrophic bacterial numbers and their association with wetland rice and its environment. Compost application in high N fertilized fields would avert the reduction of N(2)-fixing bacterial numbers and their association was beneficial to the growth of rice plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibitory effect of high N fertilization on diazotrophic bacterial numbers could be reduced by the application of compost and this observation would encourage more usage of organic manure. This study has also thrown light on the wider geographic distribution of G. diazotrophicus with wetland rice in temperate region where sugarcane (from which this bacterium was first reported to be associating and thereon from other plant species) is not cultivated.     

Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils

Agricultural soils are heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial and free-living nitrogen-fixing communities in soils from the Nafferton Factorial Systems Comparison (NFSC) study in northeast England were examined. The field experiment was factorial in design, with organic versus conventional crop rotation, crop protection, and fertility management factors. Soils were sampled on three dates (March, June, and September) in 2007. Total RNA was extracted from all soil samples and reverse transcribed. Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were used to analyze nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community, respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P ≤ 0.001). On all three dates, nifH activity was higher in the conventional crop rotation. In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionally fertilized plots in June (P = 0.0324) and in plots with organic crop protection in September (P = 0.0143). To our knowledge, the effects of organic and conventional farming systems on free-living diazotrophs have never been studied. An increased understanding of the impacts of management practices on free-living N fixers could allow modifications in soil management practices to optimize the activity of these organisms.     

Biological nitrogen fixation in a post-volcanic chronosequence from south-central Chile

Biological nitrogen fixation is a key ecosystem function incorporating new nitrogen (N) during primary successions. Increasing evidence from tropical and northern temperate forests shows that phosphorus (P) and molybdenum (Mo) either alone or in combination limit the activity of free-living diazotrophs. In this study, we evaluated the effects of Mo, P, and carbon (C) addition, either singly or in combination, and moisture, on diazotrophic activity in a post-volcanic forest chronosequence in south-fentral Chile. Diazotrophic activity, both free-living (associated with fine litter) and symbiotic (associated with the moss Racomitrium lanuginosum and the cyanolichens Pseudocyphellaria berberina and P. coriifolia), was evaluated by incubation of samples and subsequent acetylene reduction assays conducted in the field and laboratory, in winter, spring and autumn of two consecutive years. Results showed that diazotrophic activity varied with the season of the year (lowest during the drier spring season), successional stage (highest in the maximal stage), and N-fixer community type (highest in symbiotic diazotrophs). In general, C+P+Mo limitation was documented for heterotrophic diazotrophs and P+Mo limitation for symbiotic diazotrophs. Limitation of diazotrophic activity was not associated with soil nutrient and C status in the chronosequence. Strong inhibition of diazotrophic activity by high N addition and by low moisture suggests that reductions in precipitation expected for south-central Chile under climate change, as well as increasing inputs of reactive N from atmospheric deposition due to increasing use of N fertilizers, may drastically alter the composition and functional role of cryptogamic assemblages in native forests.     

Effect of Abandonment on Diversity and Abundance of Free-Living Nitrogen-Fixing Bacteria and Total Bacteria in the Cropland Soils of Hulun Buir,Inner Mongolia

In Inner Mongolia, steppe grasslands face desertification or degradation because of human over activity. One of the reasons for this condition is that croplands have been abandoned after inappropriate agricultural management. The soils in these croplands present heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, we assessed the molecular ecology of total and free-living nitrogen-fixing bacterial communities in soils from steppe grasslands and croplands that were abandoned for different periods (1, 5, and 25 years) and compared the degree of recovery. The abandoned croplands included in the study were natural restoration areas without human activity. Denaturing gradient gel electrophoresis and quantitative PCR (qPCR) were used to analyze the nifH and 16S rRNA genes to study free-living diazotrophs and the total bacterial community, respectively. The diversities of free-living nitrogen fixers and total bacteria were significantly different between each site (P<0.001). Neither the total bacteria nor nifH gene community structure of a cropland abandoned for 25 years was significantly different from those of steppe grasslands. In contrast, results of qPCR analysis of free-living nitrogen fixers and total bacteria showed significantly high abundance levels in steppe grassland (P<0.01 and P<0.03, respectively). In this study, the microbial communities and their gene abundances were assessed in croplands that had been abandoned for different periods. An understanding of how environmental factors and changes in microbial communities affect abandoned croplands could aid in appropriate soil management to optimize the structures of soil microorganisms.     

Differentiations of determinants for the community compositions of bacteria,fungi, and nitrogen fixers in various steppes

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Endophytic Establishment of Diazotrophic Bacteria in Auxin-Induced Tumors of Cereal Crops

Gramineous crops such as wheat (triticum oestivum), maize (zea mays), and rice (oryza sativa) develop tumorous structures (para-nodules) along primary and secondary roots when treated with low concentrations of various auxins. Rice forms additional tumors along its hypocotyle. Histologically, auxin-induced tumors appear as cancerous grown out root meristems and thus are comparable in origin and structure to stem nodules of the legume sesbania rostrata. Auxin-affected root meristems do not recover and develop further to large nodule-like organs. Introduced diazotrophs (Azospirillum spp., Azorhizobium caulinodans, Rhizobium spp.) potentially inhabit tissues of both stem and root tumors with the central meristem as a major colonization niche. Evidence is given that infecting bacteria follow a ‘crack entry’ invasion at sites where developing tumors have emerged through the root cortex and epidermis. Bacteria are shown to establish with high cell numbers inside intercellular spaces of cortical and meristematic tissues. Plant-cell infection of tumor cells takes place with bacteria found inside the cell-cytoplasm surrounded by membrane-like structures. Once inhabiting induced tumor tissues introduced diazotrophs colonize endophytically with high cell numbers. Mutant, ammonium-excreting and thus ecologically disadvantaged A. brasilense is shown to survive inside para-nodulating maize and rice plants with a dense population. Micro-aerobic nitrogenase activities of tumor inhabiting diazotrophic bacteria (A. brasilense, Azotobacter vinelandii, A. caulonidans) are in general highly increased when compared with untreated control plants. Additionally, bacterial nitrogenase activity is less sensitive to an increased oxygen tension in the root environment. The host plants benefit from the enhanced nitrogen fixation in their para-nodulating roots. Highest rates of incorporation of fixed nitrogen into host plant material is reported for para-nodule inhabiting ammonium excreting A. brasilense strain C3. The host plant potentially stimulates the nitrogenase activity of endophytically colonizing diazotrophs by providing energy in the form of a suitable carbon source. In conclusion, it is demonstrated that gramineous plants are potentially capable of developing an endophytical diazotrophic symbiosis through para-nodule formation.     

The effects of pesticides on bacterial nitrogen fixers in peanut-growing area

In the peanut production, the applications of herbicides and fungicides are a common practice. In this work, studies done under field conditions demonstrated that pesticides affected negatively the number and nitrogenase activity of diazotrophic populations of soil. Agrochemical effects were not transient, since these parameters were not recovered to pre-treatment levels even 1 year after pesticides application. Results obtained from greenhouse experiments revealed that the addition of herbicide or fungicides diminished the free-living diazotrophs number reaching levels found in soil amended with the pesticides and that the number of symbiotic diazotrophs was not affected by the insecticide assayed. The soil nitrogenase activity was not affected by fungicides and glyphosate. The effect of pesticides on the nitrogen-fixing bacteria diversity was evaluated both in field and greenhouse experiments. Analysis of clone libraries generated from the amplification of soil nifH gene showed a diminution in the genetic diversity of this bacterial community.     

Microbial communities and diazotrophic activity differ in the root‐zone of Alamo and Dacotah switchgrass feedstocks

Nitrogen (N) bioavailability is a primary limiting nutrient for crop and feedstock productivity. Associative nitrogen fixation (ANF) by diazotrophic bacteria in root‐zone soil microbial communities have been shown to provide significant amounts of N to some tropical grasses, but this potential in switchgrass, a warm‐season, temperate, US native, perennial tallgrass has not been widely studied. ‘Alamo’ and ‘Dacotah’ are cultivars of switchgrass, adapted to the southern and northern regions of the United States, respectively, and offer an opportunity to better describe this plant–bacterial association. The nitrogenase enzyme activity, microbial communities, and amino acid profiles in the root‐zones of the two ecotypes were studied at three different plant growth stages. Differences in the nitrogenase enzyme activity and free soluble amino acid profiles indicated the potential for greater nitrogen fixation in the high productivity Alamo compared with the lower productivity Dacotah. Changes in the amino acid profiles and microbial community structure (rRNA genes) of the root‐zone suggest different plant–bacterial interactions can help to explain differences in nitrogenase activity. PICRUSt analysis revealed functional differences, especially nitrogen metabolism, that supported ecotype differences in root‐zone nitrogenase enzyme activity. It is thought that the greater productivity of Alamo increased the belowground flow of carbon into roots and root‐zone habitats, which in turn support the high energy demands needed to support nitrogen fixation. Further research is thus needed to understand plant ecotype and cultivar trait differences that can be used to breed or genetically modify crop plants to support root‐zone associations with diazotrophs.     

Corals form characteristic associations with symbiotic nitrogen-fixing bacteria

The complex symbiotic relationship between corals and their dinoflagellate partner Symbiodinium is believed to be sustained through close associations with mutualistic bacterial communities, though little is known about coral associations with bacterial groups able to fix nitrogen (diazotrophs). In this study, we investigated the diversity of diazotrophic bacterial communities associated with three common coral species (Acropora millepora, Acropora muricata, and Pocillopora damicormis) from three midshelf locations of the Great Barrier Reef (GBR) by profiling the conserved subunit of the nifH gene, which encodes the dinitrogenase iron protein. Comparisons of diazotrophic community diversity among coral tissue and mucus microenvironments and the surrounding seawater revealed that corals harbor diverse nifH phylotypes that differ between tissue and mucus microhabitats. Coral mucus nifH sequences displayed high heterogeneity, and many bacterial groups overlapped with those found in seawater. Moreover, coral mucus diazotrophs were specific neither to coral species nor to reef location, reflecting the ephemeral nature of coral mucus. In contrast, the dominant diazotrophic bacteria in tissue samples differed among coral species, with differences remaining consistent at all three reefs, indicating that coral-diazotroph associations are species specific. Notably, dominant diazotrophs for all coral species were closely related to the bacterial group rhizobia, which represented 71% of the total sequences retrieved from tissue samples. The species specificity of coral-diazotroph associations further supports the coral holobiont model that bacterial groups associated with corals are conserved. Our results suggest that, as in terrestrial plants, rhizobia have developed a mutualistic relationship with corals and may contribute fixed nitrogen to Symbiodinium.     

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.     

Short-Term Response of Soil Bacteria to Carbon Enrichment in Different Soil Microsites

The response of bacteria in bulk soil and earthworm casts to carbon enrichment was studied by an RNA stable-isotope probing/terminal restriction fragment length polymorphism strategy with ¹³C-labeled glucose and acetate. Both the soil microsite status and the carbon enrichment selected rapidly for different active bacterial communities, which resulted in different degradation kinetics. Our study clearly illustrates the biases that are generated by adding C substrates to detect metabolically active bacteria in soil.     

Edaphic correlates of feedstock‐associated diazotroph communities

Miscanthus × giganteus and Panicum virgatum are potential promising bioenergy feedstock crops suitable for the temperate zone. The energy efficiency and sustainability of bioenergy production could be improved by reducing their fertilizer inputs – particularly energy intensive nitrogen fertilizers. Miscanthus is known to benefit from nitrogen fixation by associative diazotrophs. However, because the effects of edaphic‐, management‐, and plant‐related factors on feedstock‐associated diazotroph communities have not yet been characterized, it is not currently possible to optimize the nitrogen contribution to feedstock crops from associated diazotroph communities. To address this critical knowledge gap, we characterized the bacterial and diazotroph communities in the rhizosphere and endophytic compartments of both species at eight research sites across Illinois. We also quantified the nifH gene abundance in the rhizosphere soil as well as a range of soil chemistry parameters at these sites. Multivariate statistical analyses revealed that diazotroph and bacterial communities in the rhizosphere varied primarily among sites, with very small differences between host species. Conversely, diazotroph and bacterial communities in the endophytic compartments differed significantly between plant species, but did not vary substantially among sites. Finally, nifH gene abundance in the rhizospheres of both species varied substantially from site to site and was positively correlated with soil iron concentration as well as soil ammonium concentration, and negatively correlated with abundance of other soil nutrients including calcium, total nitrogen, and nitrates. These results indicate the potential edaphic drivers of associative diazotroph communities in feedstock rhizospheres and suggest that manipulating bioavailable iron content in the soil is a potential direction for investigating the optimization of these communities to improve their nitrogen contribution to crops.     

Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop

Aims

Previous studies have shown that elephant grass is colonized by nitrogen-fixing bacterial species; however, these results were based on culture-dependent methods, an approach that introduces bias due to an incomplete assessment of the microbial community. In this study, we used culture-independent methods to survey the diversity of endophytes and plant-associated bacterial communities in five elephant grass genotypes used in bioenergy production.

Methods

The plants of five genotypes of elephant grass were harvested from the experimental area of Embrapa Agrobiologia and divided into stem and root tissues. Total DNA and RNA were extracted from plant tissues and the bacterial communities were analyzed by DGGE and clone library of the 16S rRNA and nifH genes at both the cDNA and DNA levels.

Results

Overall, the patterns based on DNA- and RNA-derived DGGE-profiles differed, especially within tissue samples. DNA-based DGGE indicated that both total bacterial and diazotrophic communities associated with roots (rhizoplane?+?endophytes) differed clearly from those obtained from stems (endophytes). These results were confirmed by the phylogenetic analyses of RNA-derived sequences of 16S rRNA (total bacteria; 586 sequences), but not for nifH (186). In fact, rarefaction analyses showed a higher diversity of diazotrophic organisms associated with stems than roots. Based on 16S rRNA sequences, the clone libraries were dominated by sequences affiliated to members of Leptotrix (12.8 %) followed by Burkholderia (9 %) and Bradyrhizobium (6.5 %), while most of the nifH clones were closely related to the genus Bradyrhizobium (26 %).

Conclusions

Our results revealed an unexpectedly large diversity of metabolically active bacteria, providing new insights into the bacterial species predominantly found in association with elephant grass. Furthermore, these results can be very useful for the development of new strategies for selection of potential bacteria that effectively contribute to biological nitrogen fixation and enhance the sustainable production of elephant grass as bioenergy crop.     

Soil type is the primary determinant of the composition of the total and active bacterial communities in arable soils

Degradation of agricultural land and the resulting loss of soil biodiversity and productivity are of great concern. Land-use management practices can be used to ameliorate such degradation. The soil bacterial communities at three separate arable farms in eastern England, with different farm management practices, were investigated by using a polyphasic approach combining traditional soil analyses, physiological analysis, and nucleic acid profiling. Organic farming did not necessarily result in elevated organic matter levels; instead, a strong association with increased nitrate availability was apparent. Ordination of the physiological (BIOLOG) data separated the soil bacterial communities into two clusters, determined by soil type. Denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism analyses of 16S ribosomal DNA identified three bacterial communities largely on the basis of soil type but with discrimination for pea cropping. Five fields from geographically distinct soils, with different cropping regimens, produced highly similar profiles. The active communities (16S rRNA) were further discriminated by farm location and, to some degree, by land-use practices. The results of this investigation indicated that soil type was the key factor determining bacterial community composition in these arable soils. Leguminous crops on particular soil types had a positive effect upon organic matter levels and resulted in small changes in the active bacterial population. The active population was therefore more indicative of short-term management changes.     

Soil Type Is the Primary Determinant of the Composition of the Total and Active Bacterial Communities in Arable Soils

Degradation of agricultural land and the resulting loss of soil biodiversity and productivity are of great concern. Land-use management practices can be used to ameliorate such degradation. The soil bacterial communities at three separate arable farms in eastern England, with different farm management practices, were investigated by using a polyphasic approach combining traditional soil analyses, physiological analysis, and nucleic acid profiling. Organic farming did not necessarily result in elevated organic matter levels; instead, a strong association with increased nitrate availability was apparent. Ordination of the physiological (BIOLOG) data separated the soil bacterial communities into two clusters, determined by soil type. Denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism analyses of 16S ribosomal DNA identified three bacterial communities largely on the basis of soil type but with discrimination for pea cropping. Five fields from geographically distinct soils, with different cropping regimens, produced highly similar profiles. The active communities (16S rRNA) were further discriminated by farm location and, to some degree, by land-use practices. The results of this investigation indicated that soil type was the key factor determining bacterial community composition in these arable soils. Leguminous crops on particular soil types had a positive effect upon organic matter levels and resulted in small changes in the active bacterial population. The active population was therefore more indicative of short-term management changes.     

High Diversity of Diazotrophs in the Forefield of a Receding Alpine Glacier

Forefields of receding glaciers are unique and sensitive environments representing natural chronosequences. In such habitats, microbial nitrogen fixation is of particular interest since the low concentration of bioavailable nitrogen is one of the key limitations for growth of plants and soil microorganisms. Asymbiotic nitrogen fixation in the Damma glacier (Swiss Central Alps) forefield soils was assessed using the acetylene reduction assay. Free-living diazotrophic diversity and population structure were resolved by assembling four NifH sequence libraries for bulk and rhizosphere soils at two soil age classes (8- and 70-year ice-free forefield). A total of 318 NifH sequences were analyzed and grouped into 45 unique phylotypes. Phylogenetic analyses revealed a higher diversity as well as a broader distribution of NifH sequences among phylogenetic clusters than formerly observed in other environments. This illustrates the importance of free-living diazotrophs and their potential contribution to the global nitrogen input in this nutrient-poor environment. NifH diversity in bulk soils was higher than in rhizosphere soils. Moreover, the four libraries displayed low similarity values. This indicated that both soil age and the presence of pioneer plants influence diversification and population structure of free-living diazotrophs.     

Analysis of the microbial community and geochemistry of a sediment core from Great Slave Lake, Canada

Sediment cores taken from Great Slave Lake, Canada, were analysed to investigate their metabolically active microbial populations and geochemistry. The amplification of cDNA detected metabolically active bacterial (50 separate bands) and archaeal (49 separate band) communities. The bacterial communities were further resolved indicating active actinobacterial and γ-proteobacterial communities (36 and 43 individual bands respectively). Redundancy discriminate analysis and Monte Carlo permutation testing demonstrated the significant impact of geochemical parameters on microbial community structures. Geochemical analyses suggest that the upper 0.4 m represents soil weathering and erosion in the lake catchment. An increase in organic carbon in the lower core suggests either more primary productivity, indicating warmer climate conditions, associated with Holocene Climatic Optimum conditions pre 5,000 years BP or change from a reducing environment in the lower core to an oxidizing environment during more recent deposition. Drivers for bacterial, archaeal and actinobacterial community structures were sediment particle size, and its mineral composition. Depth also significantly affected γ- proteobacterial community structure. In contrast the organic carbon content did not significantly shape the microbial community structures within the sediment. This study indicates that geochemical parameters significantly contribute to microbial community structure in these sediments.