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.     

Comparison of Rhizosphere Bacterial Communities in Arabidopsis thaliana Mutants for Systemic Acquired Resistance

Systemic acquired resistance (SAR) is an inducible systemic plant defense against a broad spectrum of plant pathogens, with the potential to secrete antimicrobial compounds into the soil. However, its impact on rhizosphere bacteria is not known. In this study, we examined fingerprints of bacterial communities in the rhizosphere of the model plant Arabidopsis thaliana to determine the effect of SAR on bacterial community structure and diversity. We compared Arabidopsis mutants that are constitutive and non-inducible for SAR and verified SAR activation by measuring pathogenesis-related protein activity via a β-glucoronidase (GUS) reporter construct driven by the β-1-3 glucanase promoter. We used terminal restriction fragment length polymorphism (T-RFLP) analysis of MspI- and HaeIII-digested 16S rDNA to estimate bacterial rhizosphere community diversity, with Lactobacillus sp. added as internal controls. T-RFLP analysis showed a clear rhizosphere effect on community structure, and diversity analysis of both rhizosphere and bulk soil operational taxonomic units (as defined by terminal restriction fragments) using richness, Shannon–Weiner, and Simpson’s diversity indices and evenness confirmed that the presence of Arabidopsis roots significantly altered bacterial communities. This effect of altered soil microbial community structure by plants was also seen upon multivariate cluster analysis of the terminal restriction fragments. We also found visible differences in the rhizosphere community fingerprints of different Arabidopsis SAR mutants; however, there was no clear decrease of rhizosphere diversity because of constitutive SAR expression. Our study suggests that SAR can alter rhizosphere bacterial communities, opening the door to further understanding and application of inducible plant defense as a driving force in structuring soil bacterial assemblages.     

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.     

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.     

Archaeal and Bacterial Communities Associated with the Surface Mucus of Caribbean Corals Differ in Their Degree of Host Specificity and Community Turnover Over Reefs

Comparative studies on the distribution of archaeal versus bacterial communities associated with the surface mucus layer of corals have rarely taken place. It has therefore remained enigmatic whether mucus-associated archaeal and bacterial communities exhibit a similar specificity towards coral hosts and whether they vary in the same fashion over spatial gradients and between reef locations. We used microbial community profiling (terminal-restriction fragment length polymorphism, T-RFLP) and clone library sequencing of the 16S rRNA gene to compare the diversity and community structure of dominant archaeal and bacterial communities associating with the mucus of three common reef-building coral species (Porites astreoides, Siderastrea siderea and Orbicella annularis) over different spatial scales on a Caribbean fringing reef. Sampling locations included three reef sites, three reef patches within each site and two depths. Reference sediment samples and ambient water were also taken for each of the 18 sampling locations resulting in a total of 239 samples. While only 41% of the bacterial operational taxonomic units (OTUs) characterized by T-RFLP were shared between mucus and the ambient water or sediment, for archaeal OTUs this percentage was 2-fold higher (78%). About half of the mucus-associated OTUs (44% and 58% of bacterial and archaeal OTUs, respectively) were shared between the three coral species. Our multivariate statistical analysis (ANOSIM, PERMANOVA and CCA) showed that while the bacterial community composition was determined by habitat (mucus, sediment or seawater), host coral species, location and spatial distance, the archaeal community composition was solely determined by the habitat. This study highlights that mucus-associated archaeal and bacterial communities differ in their degree of community turnover over reefs and in their host-specificity.     

Positive correlation between soil bacterial metabolic and plant species diversity and bacterial and fungal diversity in a vegetation succession on Karst

The hypothesis that positive links exist among plant taxonomic diversity, belowground microbial taxonomic and metabolic diversities was tested for four secondary vegetation successional stages (tussock (T), shrub (S), secondary forest (SF) and primary forest (PF)) in Huanjiang county, SW China. Soil bacterial communities were characterized by DNA fingerprinting and metabolic profiling. Along the succession, Shannon diversity indices followed the order SF>PF>S>T for plant taxonomic diversity, T>SF>PF>S for bacterial operational taxonomic diversity, SF>T>S>PF for fungal operational taxonomic diversity, and SF>PF>S>T for bacterial metabolic diversity. Significant positive correlations were found between bacterial and fungal taxonomic diversities. However, there was no significant correlation between soil microbial taxonomic diversity and bacterial metabolic diversity. Two-way ANOVA revealed that vegetation and season, as well as their interaction, had significant effects on soil microbial (fungal and bacterial) taxonomic diversities, but that there were no seasonal effects on metabolic diversity. However, PCA and MANOVA revealed highly significant differences among the bacterial community-level physiological profiles, reflecting the successional sequence. The findings from this survey support the notion that there are strong interactions between aboveground and belowground communities and suggest that bacterial metabolic and plant taxonomic diversities, but not microbial taxonomic and metabolic diversities, can be correlated.     

Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.     

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.     

Bacterial communities associated with the ctenophores Mnemiopsis leidyi and Beroe ovata

Residing in a phylum of their own, ctenophores are gelatinous zooplankton that drift through the ocean's water column. Although ctenophores are known to be parasitized by a variety of eukaryotes, no studies have examined their bacterial associates. This study describes the bacterial communities associated with the lobate ctenophore Mnemiopsis leidyi and its natural predator Beroe ovata in Tampa Bay, Florida, USA. Investigations using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes demonstrated that ctenophore bacterial communities were distinct from the surrounding water. In addition, each ctenophore genus contained a unique microbiota. Ctenophore samples contained fewer bacterial operational taxonomic units (OTUs) by T-RFLP and lower diversity communities by 16S rRNA gene sequencing than the water column. Both ctenophore genera contained sequences related to bacteria previously described in marine invertebrates, and sequences similar to a sea anemone pathogen were abundant in B.?ovata. Temporal sampling revealed that the ctenophore-associated bacterial communities varied over time, with no single OTU detected at all time points. This is the first report of distinct and dynamic bacterial communities associated with ctenophores, suggesting that these microbial consortia may play important roles in ctenophore ecology. Future work needs to elucidate the functional roles and mode of acquisition of these bacteria.     

Molecular analysis of bacterial communities associated with the roots of Douglas fir (Pseudotsuga menziesii) colonized by different ectomycorrhizal fungi

We studied the effect of ectomycorrhizal fungi on bacterial communities colonizing roots of Douglas fir (Pseudotsuga menziesii). Mycorrhizal tips were cleaned of soil and separated based on gross morphological characteristics. Sequencing of the internal transcribed spacers of the nuclear rRNA gene cluster indicated that the majority of the tips were colonized by fungi in the Russulaceae, with the genera Russula and Lactarius comprising 70% of the tips. Because coamplification of organellar 16S rRNA genes can interfere with bacterial community analysis of root tips, we developed and tested a new primer pair that permits amplification of bacterial 16S rRNA genes but discriminates more effectively against organellar sequences than commonly used bacterial primer sets. We then used terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of the 16S rRNA gene to examine differences in bacterial communities associated with the mycorrhizal tips. Cluster analysis of T-RFLP profiles indicated that there were different bacterial communities among the root tips; however, the communities did not seem to be affected by the taxonomic identity of the ectomycorrhizal fungi. Terminal restriction fragment profiling and sequencing of cloned partial 16S rRNA genes indicated that most bacteria on the ectomycorrhizal tips were related to the Alphaproteobacteria and the Bacteroidetes group.     

Characterization of microbial communities found in the human vagina by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.     

Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Diversity of aquatic prokaryotic communities in the Cuatro Cienegas basin

The Cuatro Cienegas basin (Coahuila, México) is a composite of different water systems in the middle of the desert with unusually high levels of endemism and diversity in different taxa. Although the diversity of macrobiota has been well described, little is known about the diversity and distribution of microorganisms in the oligotrophic ponds. Here we describe the extent and distribution of diversity found in aquatic prokaryotic communities by analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes and phylogenetic analysis of cloned genes. Twelve locations within the basin were sampled. Among all the samples, we found a total of 117 operational taxonomic units (OTUs) using T-RFLPs, which ranged in any single sample from four to 49. OTU richness and Shannon diversity indices for different sites varied, but none were particularly high. 16S rRNA gene sequence data showed 68 different phylotypes among 198 clones. The most abundant phylotypes were Gamma- and Betaproteobacteria, and extreme halophiles. The differences among sites were significant; 45 TRFs were found only once, and 37% of the total diversity was represented by differences between sites, suggesting high beta-diversity. Further studies are needed to test whether this is a direct consequence of environmental heterogeneity in the basin.     

Response of Estuarine Biofilm Microbial Community Development to Changes in Dissolved Oxygen and Nutrient Concentrations

The information content and responsiveness of microbial biofilm community structure, as an integrative indicator of water quality, was assessed against short-term changes in oxygen and nutrient loading in an open-water estuarine setting. Biofilms were grown for 7-day periods on artificial substrates in the Pensacola Bay estuary, Florida, in the vicinity of a wastewater treatment plant (WWTP) outfall and a nearby reference site. Substrates were deployed floating at the surface and near the benthos in 5.4 m of water. Three sampling events covered a 1-month period coincident with declining seasonal WWTP flow and increasing dissolved oxygen (DO) levels in the bottom waters. Biomass accumulation in benthic biofilms appeared to be controlled by oxygen rather than nutrients. The overriding effect of DO was also seen in DNA fingerprints of community structure by terminal restriction fragment length polymorphism (T-RFLP) of amplified 16S rRNA genes. Ribotype diversity in benthic biofilms at both sites dramatically increased during the transition from hypoxic to normoxic. Terminal restriction fragment length polymorphism patterns showed pronounced differences between benthic and surface biofilm communities from the same site in terms of signal type, strength, and diversity, but minor differences between sites. Sequencing of 16S rRNA gene clone libraries from benthic biofilms at the WWTP site suggested that low DO levels favored sulfate-reducing prokaryotes (SRP), which decreased with rising oxygen levels and increasing overall diversity. A 91-bp ribotype in the CfoI-restricted 16S rRNA gene T-RFLP profiles, indicative of SRP, tracked the decrease in relative SRP abundance over time.     

Terminal restriction fragment length polymorphism analysis for human fecal microbiota and its application for analysis of complex bifidobacterial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T-RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host-specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T-RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus-specific PCR and T-RFLP could identify more than one bifidobacterial species. T-RFLP patterns of Bifidobacterium genus-specific PCR products from the fecal samples were host-specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium species- and group-specific PCR. Our study demonstrates that T-RFLP analysis is useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, and that the applied method is useful for rapid and sensitive analysis of bifidobacterial community.     

Impact of flooding on soil bacterial communities associated with poplar (Populus sp.) trees

Soil bacterial communities were analyzed in different habitats (bulk soil, rhizosphere, rhizoplane) of poplar tree microcosms (Populus tremulaxP. alba) using cultivation-independent methods. The roots of poplar trees regularly experience flooded and anoxic conditions. Therefore, we also determined the effect of flooding on microbial communities in microcosm experiments. Total community DNA was extracted and bacterial 16S rRNA genes were amplified by PCR and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and sequencing. Clone libraries were created from all three habitats under both unflooded and flooded conditions resulting in a total of 281 sequences. Numbers of different sequences (<97% similarity) in the different habitats represented 16-55% of total bacterial species richness determined from the nonparametric richness estimator Chao1. According to the number of different terminal restriction fragments (T-RFs), all of the different habitats contained approximately 20 different operational taxonomic units (OTUs), except the flooded rhizoplane habitat whose community contained less OTUs. Results of cloning and T-RFLP analysis generally supported each other. Correspondence analysis of T-RFLP patterns showed that the bacterial communities were different in bulk soil, rhizosphere and rhizoplane and changed upon flooding. For example OTUs representing Bacillus sp. were highest in the unflooded bulk soil and rhizosphere. Sequences related to Aquaspirillum, in contrast, were predominant on the poplar roots and in the rhizosphere of flooded microcosms but were rarely found in the other habitats.     

Archaea, Bacteria, and Algal Plastids Associated with the Reef-Building Corals Siderastrea stellata and Mussismilia hispida from Búzios, South Atlantic Ocean, Brazil

Reef-building corals may be seen as holobiont organisms, presenting diverse associated microbial communities. Best known is the symbiotic relationship with zooxanthellae, but Archaea, Bacteria, fungi, viruses, and algal plastids are also abundant. Until now, there is little information concerning microbial communities associated with Brazilian corals. The present study aims to describe the diversity of Archaea, Bacteria, and eukaryotic algal plastid communities associated with two sympatric species, Siderastrea stellata and Mussismilia hispida, from Southeastern Brazil, using 16S rRNA gene libraries. Since corals present a high number of other associated invertebrates, coral barcoding (COI) was performed to confirm the exclusive occurrence of coral DNA in our samples. Our analysis yielded 354 distinct microbial OTUs, represented mainly by novel phylotypes. Richness (Chao1 and ACE) and diversity (H') estimations of the microbial communities associated with both species were high and comparable to other studies. Rarefaction analyses showed that microbial diversity of S. stellata is higher than that of M. hispida. Libshuff comparative analyses showed that the highest microbial community similarity between the two coral species occurred in the bacterial libraries, while archaeal and plastidial communities were significantly different. Crenarchaeota dominated archaeal communities, while Proteobacteria was the most abundant bacterial phylum, dominated by alpha-Proteobacteria. Plastids were also represented by novel phylotypes and did not match with any 16S rRNA sequences of Cyanobacteria and zooxanthellae from GenBank. Our data improves the pool of available information on Brazilian coral microbes and shows corals as sources of diverse prokaryotic and picoeukaryotic communities.