Comparative study of endophytic and endophytic diazotrophic bacterial communities across rice landraces grown in the highlands of northern Thailand

Communities of bacterial endophytes within the rice landraces cultivated in the highlands of northern Thailand were studied using fingerprinting data of 16S rRNA and nifH genes profiling by polymerase chain reaction–denaturing gradient gel electrophoresis. The bacterial communities’ richness, diversity index, evenness, and stability were varied depending on the plant tissues, stages of growth, and rice cultivars. These indices for the endophytic diazotrophic bacteria within the landrace rice Bue Wah Bo were significantly the lowest. The endophytic bacteria revealed greater diversity by cluster analysis with seven clusters compared to the endophytic diazotrophic bacteria (three clusters). Principal component analysis suggested that the endophytic bacteria showed that the community structures across the rice landraces had a higher stability than those of the endophytic diazotrophic bacteria. Uncultured bacteria were found dominantly in both bacterial communities, while higher generic varieties were observed in the endophytic diazotrophic bacterial community. These differences in bacterial communities might be influenced either by genetic variation in the rice landraces or the rice cultivation system, where the nitrogen input affects the endophytic diazotrophic bacterial community.     

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.     

Diversity of free-living nitrogen-fixing microorganisms in the rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings

The composition of free-living nitrogen-fixing microbial communities in rhizosphere and non-rhizosphere of pioneer plants growing on wastelands of copper mine tailings was studied by the presence of nifH genes using Polymerase Chain Reaction-Denatured Gradient Gel Electrophoresis (PCR-DGGE) approach. Eleven rhizosphere tailing samples and nine non-rhizosphere tailing samples from six plant communities were collected from two wastelands with different discarded periods. The nested PCR method was used to amplify the nifH genes from environmental DNA extracted from tailing samples. Twenty-two of 37 nifH gene sequences retrieved from DGGE gels clustered in Proteobacteria (α-Proteobacteria and β-Proteobacteria) and 15 nifH gene sequences in Cyanobacteria. Most nifH gene fragments sequenced were closely related to uncultured bacteria and cyanobacteria and exhibited less than 90% nucleotide acid identity with bacteria in the database, suggesting that the nifH gene fragments detected in copper mine tailings may represent novel sequences of nitrogen-fixers. Our results indicated that the non-rhizosphere tailings generally presented higher diversity of nitrogen-fixers than rhizosphere tailings and the diversity of free-living nitrogen-fixers in tailing samples was mainly affected by the physico-chemical properties of the wastelands and plant species, especially the changes of nutrient and heavy metal contents caused by the colonization of plant community.     

Community structure analyses are more sensitive to differences in soil bacterial communities than anonymous diversity indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling

Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used for profiling microbial communities in various environments. However, it may suffer from biases during the analytic process. This study addressed the potential of T-RFLP profiles (1) to reflect real community structures and diversities, as well as (2) to reliably detect changing components of microbial community structures. For this purpose, defined artificial communities of 30 SSU rRNA gene clones, derived from nine bacterial phyla, were used. PCR amplification efficiency was one primary bias with a maximum variability factor of 3.5 among clones. PCR downstream analyses such as enzymatic restriction and capillary electrophoresis introduced a maximum bias factor of 4 to terminal restriction fragment (T-RF) signal intensities, resulting in a total maximum bias factor of 14 in the final T-RFLP profiles. In addition, the quotient between amplification efficiency and T-RF size allowed predicting T-RF abundances in the profiles with high accuracy. Although these biases impaired detection of real community structures, the relative changes in structures and diversities were reliably reflected in the T-RFLP profiles. These data support the suitability of T-RFLP profiling for monitoring effects on microbial communities.     

Spatial-temporal variability in diazotroph assemblages in Chesapeake Bay using an oligonucleotide nifH microarray

The distribution of nitrogen-fixing microorganisms in the Chesapeake Bay was investigated using fingerprints from a nifH microarray comprised of 706 60-mer oligonucleotide nifH probes representing cultivated organisms and environmental clones from different nifH clusters. Diverse nifH targets, amplified from samples using degenerate nifH primers, were detected in water column and sediment samples collected in April and October, 2001-2002. Total nifH richness and diversity (Simpson's and Shannon indices) were highest at the most riverine, oligohaline North Bay station. In most samples, the highest diversity was in nifH Cluster 3, which includes many anaerobes, while Cluster 1 (alpha-, beta- gamma- Proteobacteria, Cyanobacteria) targets had the greatest microarray signal intensities. In a multidimensional scaling analysis, deep water communities from April and October were similar within each of the sampling sites, while the surface communities had more variability. Diazotroph communities in the water column in the North Bay were distinct from the Mid- and South Bay communities, and there was a gradual change in sediment diazotroph assemblages from the North to the South Bay. Diazotrophic assemblages from the majority of the water column samples from the Mid- and South Bay clustered with the sediment assemblage in Mid-Bay. Dissolved inorganic nitrogen, salinity, dissolved organic carbon and dissolved organic phosphorus had a significant relationship with the diazotrophic bacterioplankton community. Higher diversity in the freshwater end of the system may reflect variability in disturbance rates and environmental conditions such as forms and concentrations of organic matter, nutrients and oxygen.     

Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol–chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.     

Seasonal Fluctuations of Bacterial Community Diversity in Agricultural Soil and Experimental Validation by Laboratory Disturbance Experiments

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.     

Application of a nifH microarray to assess the impact of environmental factors on free-living diazotrophs in a glacier forefield

Glacier forefield environments are exposed to extreme and fluctuating climatic and nutritional conditions. The high diversity of free-living diazotrophic communities found in these environments indicates that nitrogen fixers are able to efficiently cope with such conditions. In this study, a nifH microarray was used to monitor changes in diazotrophic populations in the field over a season, in the presence or absence of plants and in 2 glacier forefields characterized by a different bedrock type (siliceous or calcareous), as well as at different temperatures (10 °C, 15 °C) and under different nitrogen fertilization regimes (0, 10, 40 kg N·ha(-1)·year(-1)) in laboratory systems. Population structures responded highly dynamically to environmental changes. Plant presence had the strongest impact, which decreased toward the end of the season and with high amounts of nitrogen fertilization. Temperature and nitrogen fertilization increases indirectly affected diazotrophic communities through their positive impact on plant growth. These results indicate strong carbon limitation in young glacier forefield soils. Phylotypes related to the genus Methylocystis strongly responded to environmental variations. These methanotrophic microorganisms, which are able to retrieve nitrogen and carbon from the atmospheric pool, are particularly adapted to the extreme nutritional conditions found in glacier forefields.     

Influence of temperature and soil water content on bacterial, archaeal and denitrifying microbial communities in drained fen grassland soil microcosms

In this study, microcosms were used to investigate the influence of temperature (4 and 28 degrees C) and water content (45% and 90% WHC) on microbial communities and activities in carbon-rich fen soil. Bacterial, archaeal and denitrifier community composition was assessed during incubation of microcosms for 12 weeks using terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA and nitrous oxide reductase (nosZ) genes. In addition, microbial and denitrifier abundance, potential denitrification activity and production of greenhouse gases were measured. No detectable changes were observed in prokaryote or denitrifier abundance. In general, cumulatively after 12 weeks more carbon was respired at the higher temperature (3.7 mg CO(2) g(-1) soil), irrespective of the water content, whereas nitrous oxide production was greater under wet conditions (98-336 microg N(2)O g(-1) soil). After an initial lag phase, methane emissions (963 microg CH(4) g(-1) soil) were observed only under warm and wet conditions. T-RFLP analyses of bacterial 16S rRNA and nosZ genes revealed small or undetectable community changes in response to temperature and water content, suggesting that bacterial and denitrifying microbial communities are stable and do not respond significantly to seasonal changes in soil conditions. In contrast, archaeal microbial community structure was more dynamic and was strongly influenced by temperature.     

Interpreting 16S rDNA T-RFLP Data: Application of Self-Organizing Maps and Principal Component Analysis to Describe Community Dynamics and Convergence

Interpreting the large amount of data generated by rapid profiling techniques, such as T-RFLP, DGGE, and DNA arrays, is a difficult problem facing microbial ecologists. This study compares the ability of two very different ordination methods, principal component analysis (PCA) and self-organizing map neural networks (SOMs), to analyze 16S-DNA terminal restriction-fragment length polymorphism (T-RFLP) profiles from microbial communities in glucose-fed methanogenic bioreactors during startup and changes in operational parameters. Our goal was not only to identify which samples were similar, but also to decipher community dynamics and describe specific phylotypes, i.e., phylogenetically similar organisms, that behaved similarly in different reactors. Fifteen samples were taken over 56 volume changes from each of two bioreactors inoculated from river sediment (S2) and anaerobic digester sludge (M3) and from a well-established control reactor (R1). PCA of bacterial T-RFLP profiles indicated that both the S2 and M3 communities changed rapidly during the first nine volume changes, and then became relatively stable. PCA also showed that an HRT of 8 or 6 days had no effect on either reactor communtity, while an HRT of 2 days changed community structure significantly in both reactors. The SOM clustered the terminal restriction fragments according to when each fragment was most abundant in a reactor community, resulting in four clearly discernible groups. Thirteen fragments behaved similarly in both reactors, eight of which composed a significant proportion of the microbial community as judged by the relative abundance of the fragment in the T-RFLP profiles. Six Bacteria terminal restriction fragments shared between the two communities matched cloned 16S rDNA sequences from the reactors related to Spirochaeta, Aminobacterium, Thermotoga, and Clostridium species. Convergence also occurred within the acetoclastic methanogen community, resulting in a predominance of Methanosarcina siciliae-related organisms. The results demonstrate that both PCA and SOM analysis are useful in the analysis of T-RFLP data; however, the SOM was better at resolving patterns in more complex and variable data than PCA ordination.     

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico

Background

Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP), might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods.

Methodology/Principal Findings

We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6%) of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil).

Conclusions/Significance

The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the ambient environment, allowed us to differentiate resident symbionts from potentially transient or prey bacteria. Pairing replicated clone library construction with rapid community profiling via T-RFLP analyses will greatly facilitate future studies of sponge-microbe symbioses.     

Quantifying functional gene populations: comparing gene abundance and corresponding enzymatic activity using denitrification and nitrogen fixation in pulp and paper mill effluent treatment systems.

The relationship between the abundance of three functional genes and their corresponding biochemical reaction rates was investigated in several activated sludge and mill effluent microbial communities. Gene probes were prepared for two key denitrification genes (nirS and nirK) and for one nitrogen-fixation gene (nifH) and were validated using a variety of strains of known nir and nif genotype. ATP-based measures of viable cell numbers were used to provide total population sizes. In certain microbial communities (activated sludge enrichment cultures and multiple samples taken from the same mill primary clarifier), a strong correlation was observed between gene abundance and biochemical activity rates. However, when comparing several different nonenriched activated sludge bioreactors and separate primary clarifier microbial communities, the ratio of specific gene abundance to biochemical activity rates varied widely. These results suggest that in cases where a microbial community is not fully induced for a given biochemical activity or when very different communities are compared, quantitative gene probing can give a better measure of a community's potential to carry out the encoded function than can the relevant biochemical assay. However, the gene quantitation method employed here probably underestimated the true number of probed genes present in the microbial communities due to nirS and nifH genes in the communities having reduced DNA sequence similarity with the probes used.     

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.     

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) is a rapid, robust, inexpensive and simple tool for microbial community profiling. Methods used for DNA extraction, PCR amplification and digestion of amplified products have a considerable impact on the results of T-RFLP. Pitfalls of the method skew the similarity analysis and compromise its high throughput ability. Despite a high throughput method of data generation, data analysis is still in its infancy and needs more attention. Current article highlights the limitations of the methods used for data generation and analysis. It also provides an overview of the recent methodological developments in T-RFLP which will assist the readers in obtaining real and authentic profiles of the microbial communities under consideration while eluding the inherent biases and technical difficulties.     

Bacterial communities in Arctic fjelds of Finnish Lapland are stable but highly pH-dependent

The seasonal and spatial variations of microbial communities in Arctic fjelds of Finnish Lapland were studied. Phospholipid fatty acid analysis (PLFA) and terminal restriction fragment analysis (T-RFLP) of amplified 16S rRNA genes were used to assess the effect of soil conditions and vegetation on microbial community structures along different altitudes of two fjelds, Saana and Jehkas. Terminal restriction fragments were additionally analysed from c. 160 cloned sequences and isolated bacterial strains and matched with those of soil DNA samples. T-RFLP and PLFA analyses indicated relatively similar microbial communities at various altitudes and under different vegetation of the two fjelds. However, soil pH had a major influence on microbial community composition. Members of the phylum Acidobacteria dominated especially in the low pH soils (pH 4.6-5.2), but above pH 5.5, the relative amount of terminal restriction fragments corresponding to acidobacterial clones was substantially lower. Both T-RFLP and PLFA analysis indicated stable microbial communities as the DNA and fatty acid profiles were similar in spring and late summer samples sampled over 3 years. These results indicate that differences in microbial community composition could be explained primarily by variation in the bedrock materials that cause variation in the soil pH.