rRNA Gene Clone Research Articles

Microbiota of a UASB Reactor Treating Palm Oil Mill Effluent Using HiSeq Sequencing

Potential microbial communities in the UASB reactor fed with two different substrates i.e synthetic wastewater and raw palm oil mill effluent (POME) were elucidated by using one of the most popular techniques in molecular methods, viz 16S rDNA cloning. The methodology involved; the extraction of nucleic acids, amplification and cloning of the 16S rRNA genes on sequencing HiSeq platform and finally identification and affiliation of the isolated clone with the aid of phylogenetic software. Results showed that the genus methanosarcina and methanosaeta were dominant methanogens in this study for both substrates types. Overall, microbial population (Bacteria and Archaea) in sample A (POME as substrate) is more diverse compared to sample B (synthetic wastewater as substrate) due to abundance of microorganism population in raw POME which was used as a substrate. However for the methanogenic (Archaea) diversity in both samples, there was not much different between sample A and sample B probably due to similar inoculum was inoculated in the reactor despite of have using different substrate type.

Study of Genetic Diversity of Micro-planktonic Eukaryotes in South China Sea by ITS and 5.8s rRNA Gene Cloning and Sequencing

Study of Genetic Diversity of Micro-planktonic Eukaryotes in South China Sea by ITS and 5.8s rRNA Gene Cloning and Sequencing

Environmental Biotechnology: Current Advances, New Knowledge Gaps, and Emerging Issues.

Environmental Biotechnology: Current Advances, New Knowledge Gaps, and Emerging Issues.

Comparative evaluation of the indigenous microbial diversity vs. drilling fluid contaminants in the NEEM Greenland ice core

Demonstrating that the detected microbial diversity in nonaseptically drilled deep ice cores is truly indigenous is challenging because of potential contamination with exogenous microbial cells. The NEEM Greenland ice core project provided a first-time opportunity to determine the origin and extent of contamination throughout drilling. We performed multiple parallel cultivation and culture-independent analyses of five decontaminated ice core samples from different depths (100-2051 m), the drilling fluid and its components Estisol and Coasol, and the drilling chips collected during drilling. We created a collection of diverse bacterial and fungal isolates (84 from the drilling fluid and its components, 45 from decontaminated ice, and 66 from drilling chips). Their categorization as contaminants or intrinsic glacial ice microorganisms was based on several criteria, including phylogenetic analyses, genomic fingerprinting, phenotypic characteristics, and presence in drilling fluid, chips, and/or ice. Firmicutes and fungi comprised the dominant group of contaminants among isolates and cloned rRNA genes. Conversely, most Proteobacteria and Actinobacteria originating from the ice were identified as intrinsic. This study provides a database of potential contaminants useful for future studies of NEEM cores and can contribute toward developing standardized protocols for contamination detection and ensuring the authenticity of the microbial diversity in deep glacial ice.

Bacterial microbiome of Coptotermes curvignathus (Isoptera: Rhinotermitidae) reflects the coevolution of species and dietary pattern

AbstractCoptotermes curvignathus Holmgren is capable of feeding on living trees. This ability is attributed to their effective digestive system that is furnished by the termite's own cellulolytic enzymes and cooperative enzymes produced by their gut microbes. In this study, the identity of an array of diverse microbes residing in the gut of C. curvignathus was revealed by sequencing the near‐full‐length 16S rRNA genes. A total of 154 bacterial phylotypes were found. The Bacteroidetes was the most abundant phylum and accounted for about 65% of the gut microbial profile. This is followed by Firmicutes, Actinobacteria, Spirochetes, Proteobacteria, TM7, Deferribacteres, Planctomycetes, Verrucomicrobia, and Termite Group 1. Based on the phylogenetic study, this symbiosis can be a result of long coevolution of gut enterotypes with the phylogenic distribution, strong selection pressure in the gut, and other speculative pressures that determine bacterial biome to follow. The phylogenetic distribution of cloned rRNA genes in the bacterial domain that was considerably different from other termite reflects the strong selection pressures in the gut where a proportional composition of gut microbiome of C. curvignathus has established. The selection pressures could be linked to the unique diet preference of C. curvignathus that profoundly feeds on living trees. The delicate gut microbiome composition may provide available nutrients to the host as well as potential protection against opportunistic pathogen.

Biological activity in expanded clay (EC) and granulated activated carbon (GAC) seawater filters

ABSTRACT Seawater filters containing expanded clay (EC) and granulated activated carbon (GAC) media were operated for 14 months. The biological activity found after concentrating the backwashed sludge was analysed by fluorescence in situ hybridisation (FISH), scanning electron microscopy (SEM) and 16 s rRNA gene cloning. FISH and SEM analyses confirmed the presence of extracellular polymeric substances derived from bacteria, and planktonic and benthic eukaryotic microorganisms were found. The bacterial community distribution examined by cloning and sequencing of the 16 s rRNA bacterial genes demonstrated that both filter populations were dominated by the Proteobacteria class, accounting for 68 and 50% of the EC and GAC filter populations, respectively, followed by Bacteroidetes (10 and 22%), Planctomycetes (6 and 10%), Verrucomicrobia (6 and 13%) and others that occurred at lesser extents ( Dominant classes observed in both EC and GAC libraries are close to seawater reverse osmosis membrane libraries, and most of them are often linked to biofilm promoters, suggesting that both types of media could act as accumulators of biofilm promoters.

Microbial Community Structure of a Leachfield Soil: Response to Intermittent Aeration and Tetracycline Addition

Soil-based wastewater treatment systems, or leachfields, rely on microbial processes for improving the quality of wastewater before it reaches the groundwater. These processes are affected by physicochemical system properties, such as O2 availability, and disturbances, such as the presence of antimicrobial compounds in wastewater. We examined the microbial community structure of leachfield mesocosms containing native soil and receiving domestic wastewater under intermittently-aerated (AIR) and unaerated (LEACH) conditions before and after dosing with tetracycline (TET). Community structure was assessed using phospholipid fatty acid analysis (PLFA), analysis of dominant phylotypes using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE), and cloning and sequencing of 16S rRNA genes. Prior to dosing, the same PLFA biomarkers were found in soil from AIR and LEACH treatments, although AIR soil had a larger active microbial population and higher concentrations for nine of 32 PLFA markers found. AIR soil also had a larger number of dominant phylotypes, most of them unique to this treatment. Dosing of mesocosms with TET had a more marked effect on AIR than LEACH soil, reducing the size of the microbial population and the number and concentration of PLFA markers. Dominant phylotypes decreased by ~15% in response to TET in both treatments, although the AIR treatment retained a higher number of phylotypes than the LEACH treatment. Fewer than 10% of clones were common to both OPEN ACCESS Water 2013, 5 506 AIR and LEACH soil, and fewer than 25% of the clones from either treatment were homologous with isolates of known genus and species. These included human pathogens, as well as bacteria involved in biogeochemical transformations of C, N, S and metals, and biodegradation of various organic contaminants. Our results show that intermittent aeration has a marked effect on the size and structure of the microbial community that develops in a native leachfield soil. In addition, there is a differential response of the microbial communities of AIR and LEACH soil to tetracycline addition which may be linked to changes in function.

Actinomycetes Diversity among rRNA Gene Clones and Cellular Isolates from Sambhar Salt Lake, India

The vertical stratification of actinomycetes diversity in Sambhar salt lake (India's largest salt lake) was investigated by using cultivable and uncultivable approaches. The isolates from cultured approaches were clustered on the basis of cultural, morphological, biochemical, and cell wall characteristics, and results were further strengthened by 16S rDNA-RFLP into five major groups. 16S rDNA sequencing of the representative isolates from each clusters was identified as belonging to Streptomyces, Actinopolyspora, Microbispora, Saccharopolyspora, and Actinoplanes genera, while culture independent group was established as Streptomyces (130 clones, 20 OTUs), Micromonospora (96 clones, 7 OTUs), Streptosporangium (79 clones, 9 OTUs), Thermomonospora (46 clones, 8 OTUs), and Dactylosporangium (58 clones, 8 OTUs). The diversity assessment using Shannon and Wiener index was found to be 1.55, 1.52, 1.55, and 1.49 from surface lake water, at depth of 1.5 m, shallow layer of water with algal population, and finally at depth of 2.5 m, respectively. We observed diversity in terms of the species richness as Streptomyces is dominant genus in both culture dependent and culture independent techniques followed by Microbispora (culture dependent methods) and Micromonospora (culture independent method) genera, respectively.

Biofouling of reverse osmosis membranes used in river water purification for drinking purposes: analysis of microbial populations

Biofouling in water treatment processes represents one of the most frequent causes of plant performance decline. Investigation of clogged membranes (reverse osmosis membranes, microfiltration membranes and ultrafiltration membranes) is generally performed on fresh membranes. In the present study, a multidisciplinary autopsy of a reverse osmosis membrane (ROM) was conducted. The membrane, which was used in sulfate-rich river water purification for drinking purposes, had become inoperative after 6 months because of biofouling and was later stored for 18 months in dry conditions before analysis. SSU rRNA gene library construction, clone sequencing, T-RFLP, light microscope, and scanning electron microscope (SEM) observations were used to identify the microorganisms present on the membrane and possibly responsible for biofouling at the time of removal. The microorganisms were mainly represented by bacteria belonging to the phylum Actinobacteria and by a single protozoan species belonging to the Lobosea group. The microbiological analysis was interpreted in the context of the treatment plant operations to hypothesize as to the possible mechanisms used by microorganisms to enter the plant and colonize the ROM surface.

Prokaryotic ultramicroforms in a Sphagnum peat bog of upper Volga catchment

The waters of small lakes located in swampy catchment areas of upper Volga contain considerable amounts of ultrasmall microbial cells that pass through 0.22-μm-pore-size filters. As shown in our previous study [1], most of these cells represent the bacterial genera Herbaspirillum, Herminiimonas, Curvibacter, and Burkholderia of the class Betaproteobacteria, as well as euryarchaea of the uncharacterized clade LDS. The aim of the present study was to investigate the possible effect of the waters draining swampy areas on the composition of the filterable microbial fraction in lakes fed by swampy catchments. To address this question molecular identification was performed of prokaryotic ultramicroforms in the peat of the ombrotrophic Sphagnum bog Obukhovskoe, located, like the lakes studied previously [1], in the Mologa-Sheksna catchment area. The number of filterable microorganisms in 1 g wet peat was 3.8 × 106 cells, or 0.5% of total microbial cell number in the peat. From the DNA of the filterable cell fraction, 100 clones of bacterial and 77 clones of archaeal 16S rRNA genes were obtained. The bacterial clone library contained 16S rRNA gene sequences representing the classes Beta- and Gammaproteobacteria (the genera Janthinobacterium and Pseudomonas, respectively) and the phylum Bacteroidetes (the genera Chryseobacterium and Epilithonimonas) and differed significantly from the clone library of bacterial ultramicroforms of lake water. By contrast, the pools of filterable archaea in bogs and lakes were essentially similar. They were represented by the euryarchaeal clade LDS and methanogens of the orders Methanobacteriales and Methanosarcinales. Additionally, the pool of filterable archaea of the bog included methanogens of the order Methanomicrobiales and representatives of the uncharacterized euryarchaeal clade RC-V (Rice Cluster V) and of the phylum Thaumarchaeota.

Gut Microbiome of the Critically Endangered New Zealand Parrot, the Kakapo (Strigops habroptilus)

The kakapo, a parrot endemic to New Zealand, is currently the focus of intense research and conservation efforts with the aim of boosting its population above the current ‘critically endangered’ status. While virtually nothing is known about the microbiology of the kakapo, given the acknowledged importance of gut-associated microbes in vertebrate nutrition and pathogen defense, it should be of great conservation value to analyze the microbes associated with kakapo. Here we describe the first study of the bacterial communities that reside within the gastrointestinal tract (GIT) of both juvenile and adult kakapo. Samples from along the GIT, taken from the choana (≈throat), crop and faeces, were subjected to 16 S rRNA gene library analysis. Phylogenetic analysis of >1000 16 S rRNA gene clones, derived from six birds, revealed low phylum-level diversity, consisting almost exclusively of Firmicutes (including lactic acid bacteria) and Gammaproteobacteria. The relative proportions of Firmicutes and Gammaproteobacteria were highly consistent among individual juveniles, irrespective of sampling location, but differed markedly among adult birds. Diversity at a finer phylogenetic resolution (i.e. operational taxonomic units (OTUs) of 99% sequence identity) was also low in all samples, with only one or two OTUs dominating each sample. These data represent the first analysis of the bacterial communities associated with the kakapo GIT, providing a baseline for further microbiological study, and facilitating conservation efforts for this unique bird.

The Phylogenetic Affiliation of an Uncultured Population of Ammonia-Oxidizing Bacteria Harboring Environmental Sequences of amoA Cluster-3

We investigated the phylogenetic diversity of ammonia-oxidizing bacteria (AOB) in Yellow Sea continental shelf sediment by the cloning and sequencing of PCR-amplified amoA and 16S rRNA genes. Phylogenetic analysis of the amoA-related clones revealed that the diversity of AOB was extremely low at the study site. The majority (92.7%) of amoA clones obtained belonged to a single cluster, environmental amoA cluster-3, the taxonomic position of which was previously unknown. Phylogenetic analysis on AOB-specific 16S rRNA gene sequences also demonstrated a very low diversity. All of the cloned 16S rRNA gene sequences comprised a single phylotype that belonged to the members of uncultured Nitrosospira cluster-1, suggesting that AOB belonging to the uncultured Nitrosospira cluster- 1 could carry amoA sequences of environmental amoA cluster-3.

Change in Bacterial Community Structure in Response to Disturbance of Natural Hardwood and Secondary Coniferous Forest Soils in Central Taiwan

Forest management often results in changes in the soil and its microbial communities. In the present study, differences in the soil bacterial community caused by forest management practices were characterized using small subunit (SSU) ribosomal RNA (rRNA) gene clone libraries. The communities were from a native hardwood forest (HWD) and two adjacent conifer plantations in a low-elevation montane, subtropical experimental forest at the Lienhuachi Experimental Forest (LHCEF) in central Taiwan. At this locality, the elevation ranges from 600 to 950 m, the mean annual precipitation is 2,200 mm, the mean annual temperature is 20.8 °C, and the soil pH is 4. The conifer forests included a Cunninghamia konishii Hay (CNH) plantation of 40 years and an old growth Calocedrus formosana (Florin) Florin (CLC) forest of 80 years. A total of 476 clones were sequenced and assigned into 12 phylogenetic groups. Proteobacteria-affiliated clones (53%) predominated in the library from HWD soils. In contrast, Acidobacteria was the most abundant phylum and comprised 39% and 57% in the CLC and CNH libraries, respectively. Similarly, the most abundant OTUs in HWD soils were greatly reduced or absent in the CLC and CNH soils. Based on several diversity indices, the numbers of abundant OTUs and singletons, and rarefaction curves, the diversity of the HWD community (0.95 in evenness and Shannon diversity indices) was somewhat less than that in the CNH soils (0.97 in evenness and Shannon diversity indices). The diversity of the community in CLC soils was intermediate. The differences in diversity among the three communities may also reflect changes in abundances of a few OTUs. The CNH forest soil community may be still in a successional phase that is only partially stabilized after 40 years. Analysis of molecular variance also revealed that the bacterial community composition of HWD soils was significantly different from CLC and CNH soils (p = 0.001). These results suggest that the disturbance of forest conversion and tree species composition are important factors influencing the soil bacterial community among three forest ecosystems in the same climate.

Structural and functional diversity of soil bacterial and fungal communities following woody plant encroachment in the southern Great Plains

In the southern Great Plains (USA), encroachment of grassland ecosystems by Prosopis glandulosa (honey mesquite) is widespread. Mesquite encroachment alters net primary productivity, enhances stores of C and N in plants and soil, and leads to increased levels of soil microbial biomass and activity. While mesquite’s impact on the biogeochemistry of the region is well established, it effects on soil microbial diversity and function are unknown. In this study, soils associated with four plant types (C3 perennial grasses, C4 midgrasses, C4 shortgrasses, and mesquite) from a mesquite-encroached mixed grass prairie were surveyed to in an attempt to characterize the structure, diversity, and functional capacity of their soil microbial communities. rRNA gene cloning and sequencing were used in conjunction with the GeoChip functional gene array to evaluate these potential differences. Mesquite soil supported increased bacterial and fungal diversity and harbored a distinct fungal community relative to other plant types. Despite differences in composition and diversity, few significant differences were detected with respect to the potential functional capacity of the soil microbial communities. These results may suggest that a high level of functional redundancy exists within the bacterial portion of the soil communities; however, given the bias of the GeoChip toward bacterial functional genes, potential functional differences among soil fungi could not be addressed. The results of this study illustrate the linkages shared between above- and belowground communities and demonstrate that soil microbial communities, and in particular soil fungi, may be altered by the process of woody plant encroachment.

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the α-, γ- and δ-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13°N.

Culture-independent analysis of bacterial diversity in a child-care facility

BackgroundChild-care facilities appear to provide daily opportunities for exposure and transmission of bacteria and viruses. However, almost nothing is known about the diversity of microbial contamination in daycare facilities or its public health implications. Recent culture-independent molecular studies of bacterial diversity in indoor environments have revealed an astonishing diversity of microorganisms, including opportunistic pathogens and many uncultured bacteria. In this study, we used culture and culture-independent methods to determine the viability and diversity of bacteria in a child-care center over a six-month period.ResultsWe sampled surface contamination on toys and furniture using sterile cotton swabs in four daycare classrooms. Bacteria were isolated on nutrient and blood agar plates, and 16S rRNA gene sequences were obtained from unique (one of a kind) colony morphologies for species identification. We also extracted DNA directly from nine representative swab samples taken over the course of the study from both toy and furniture surfaces, and used "universal" 16S rRNA gene bacterial primers to create PCR-based clone libraries. The rRNA gene clones were sequenced, and the sequences were compared with related sequences in GenBank and subjected to phylogenetic analyses to determine their evolutionary relationships. Culturing methods identified viable bacteria on all toys and furniture surfaces sampled in the study. Bacillus spp. were the most commonly cultured bacteria, followed by Staphylococcus spp., and Microbacterium spp. Culture-independent methods based on 16S rRNA gene sequencing, on the other hand, revealed an entirely new dimension of microbial diversity, including an estimated 190 bacterial species from 15 bacterial divisions. Sequence comparisons and phylogenetic analyses determined that the clone libraries were dominated by a diverse set of sequences related to Pseudomonas spp., as well as uncultured bacteria originally identified on human vaginal epithelium. Other sequences were related to uncultured bacteria from wastewater sludge, and many human-associated bacteria including a number of pathogens and opportunistic pathogens. Our results suggest that the child-care facility provided an excellent habitat for slime-producing Pseudomonads, and that diaper changing contributed significantly to the bacterial contamination.ConclusionThe combination of culture and culture-independent methods provided powerful means for determining both viability and diversity of bacteria in child-care facilities. Our results provided insight into the source of contamination and suggested ways in which sanitation might be improved. Although our study identified a remarkable array of microbial diversity present in a single daycare, it also revealed just how little we comprehend the true extent of microbial diversity in daycare centers or other indoor environments.

Cultivation-dependent and cultivation-independent characterization of the microbial community in acid mine drainage associated with acidic Pb/Zn mine tailings at Lechang, Guangdong, China

Cultivation-based and molecular approaches were used to characterize the phylogenetic composition and structure of the microbial community in an extremely acidic (pH 2.0) acid mine drainage (AMD) associated with Pb/Zn mine tailings that were undergoing vigorous acid generation. Acidophilic bacteria were isolated and enumerated on solid media, and were found to be restricted to isolates related to Acidithiobacillus ferrooxidans and Acidiphilium cryptum. By contrast, cloning and phylogenetic analysis of 16S rRNA genes revealed that, although low in total taxonomically distinct groups, the tailings AMD ecosystem harbored a wide range of phylogenetically diverse microbes. Of the 141 clones examined, 104 were phylogenetically affiliated with the recently discovered, iron-oxidizing Leptospirillum group III within the Nitrospira. It thus appears that iron serves as the major electron donor in this habitat. Thirty clones were affiliated with the Proteobacteria, half of which belonged to organisms related to Alphaproteobacteria species capable of ferric iron reduction. Other clones were grouped with Betaproteobacteria and Gammaproteobacteria (six clones each), and even with Deltaproteobacteria (three clones), a subdivision with anaerobic sulfate or metal (iron) reduction as the predominant physiological trait of its members. Finally, four clones were clustered within the Firmicutes and the Acidobacteria. Approximately half of the sequence types representing the majority of the total clones fell into lineages that are poorly represented by cultured organisms or have thus far been represented by only a few environmental sequences. Thus, the present study extends our knowledge of the biodiversity of microorganisms populating highly acidic AMD environments.

Bacterial Community Structure on Membrane Surface and Characteristics of Strains Isolated from Membrane Surface in Submerged Membrane Bioreactor

In order to investigate the bacterial community structure and the characteristics of bacteria on the membrane surface, a submerged membrane bioreactor treating municipal wastewater was continuously operated under two different conditions. Bacterial community structures were examined by PCR‐denaturing gradient gel electrophoresis and PCR cloning of 16S rRNA genes. Bacterial strains isolated from membrane surface were identified and their growth curve, EPS concentration and hydrophobicity were measured. The structures of bacterial communities in the suspended solids and on the membrane surface were obviously different, and γ‐Proteobacteria more selectively adhere and grow on the membrane surface than other microorganisms. Most of the membrane isolates grew slowly as compared with the strains isolated from the suspended solids. Also, the membrane isolates were higher cell surface hydrophobicities, higher EPS concentrations, and higher ratios of protein to carbohydrate within the EPSs than the isolates from suspended solids.

Uncultured Archaea in a hydrothermal microbial assemblage: phylogenetic diversity and characterization of a genome fragment from a euryarchaeote

The polychaete Alvinella pompejana lives in organic tubes on the walls of active hydrothermal chimneys along the East Pacific Rise. To examine the diversity of the archaeal community associated with the polychaete tubes, we constructed libraries by direct PCR amplification and cloning of 16S rRNA genes. Almost half of the sequences of the 16S rRNA gene libraries clustered with uncultured archaeal groups. In an effort to access genomic information from uncultured archaeal members we further constructed a fosmid library from the same DNA source. One of the clones, Alv-FOS5, was sequenced completely. Its sequence analysis revealed an incomplete rRNA operon and 32 predicted ORFs. Seventeen of these ORFs have been assigned putative functions, including transcription and translation, cellular processes and signalling, transport systems and metabolic pathways. Phylogenetic analyses of the 16S rRNA gene suggested that Alv-FOS5 formed a new lineage related to members of Deep-Sea Hydrothermal Vent Euryarchaeota group II. Phylogenetic analyses of predicted proteins revealed the existence of likely cases of horizontal gene transfer, both between Crenarchaeota and Euryarchaeota and between Archaea and Bacteria. This study is the first step in using genomics to reveal the physiology of an as yet uncultured group of archaea from deep-sea hydrothermal vents.

Molecular profiling of rhizosphere microbial communities associated with healthy and diseased black spruce (Picea mariana) seedlings grown in a nursery.

Bacterial and fungal populations associated with the rhizosphere of healthy black spruce (Picea mariana) seedlings and seedlings with symptoms of root rot were characterized by cloned rRNA gene sequence analysis. Triplicate bacterial and fungal rRNA gene libraries were constructed, and 600 clones were analyzed by amplified ribosomal DNA restriction analysis and grouped into operational taxonomical units (OTUs). A total of 84 different bacterial and 31 different fungal OTUs were obtained and sequenced. Phylogenetic analyses indicated that the different OTUs belonged to a wide range of bacterial and fungal taxa. For both groups, pairwise comparisons revealed that there was greater similarity between replicate libraries from each treatment than between libraries from different treatments. Significant differences between pooled triplicate samples from libraries of genes from healthy seedlings and pooled triplicate samples from libraries of genes from diseased seedlings were also obtained for both bacteria and fungi, clearly indicating that the rhizosphere-associated bacterial and fungal communities of healthy and diseased P. mariana seedlings were different. The communities associated with healthy and diseased seedlings also showed distinct ecological parameters as indicated by the calculated diversity, dominance, and evenness indices. Among the main differences observed at the community level, there was a higher proportion of Acidobacteria, Gammaproteobacteria, and Homobasidiomycetes clones associated with healthy seedlings, while the diseased-seedling rhizosphere harbored a higher proportion of Actinobacteria, Sordariomycetes, and environmental clones. The methodological approach described in this study appears promising for targeting potential rhizosphere-competent biological control agents against root rot diseases occurring in conifer nurseries.