ENVIRONMENTAL MICROBIOLOGY, Issue 9 2010
Franck Lejzerowicz
Summary Environmental SSU rDNA-based surveys are contributing to the dramatic revision of eukaryotic high-level diversity and phylogeny as the number of sequence data increases. This ongoing revolution gives the opportunity to test for the presence of some eukaryotic taxa in environments where they have not been found using classical microscopic observations. Here, we test whether the foraminifera, a group of single-celled eukaryotes, considered generally as typical for the marine ecosystems are present in soil. We performed foraminiferal-specific nested PCR on 20 soil DNA samples collected in contrasted environments. Unexpectedly, we found that the majority of the samples contain foraminiferal SSU rDNA sequences. In total, we obtained 49 sequences from 17 localities. Phylogenetic analysis clusters them in four groups branching among the radiation of early foraminiferal lineages. Three of these groups also include sequences originated from previous freshwater surveys, suggesting that there were up to four independent colonization events of terrestrial and/or freshwater ecosystems by ancestral foraminifera. As shown by our data, foraminifera are a widespread and diverse component of soil microbial communities. Yet, identification of terrestrial foraminiferal species and understanding of their ecological role represent an exciting challenge for future research. [source]

Eukaryotic diversity and phylogeny using small- and large-subunit ribosomal RNA genes from environmental samples

ENVIRONMENTAL MICROBIOLOGY, Issue 12 2009
William Marande
Summary The recent introduction of molecular techniques in eukaryotic microbial diversity studies, in particular those based in the amplification and sequencing of small-subunit ribosomal DNA (SSU rDNA), has revealed the existence of an unexpected variety of new phylotypes. The taxonomic ascription of the organisms bearing those sequences is generally deduced from phylogenetic analysis. Unfortunately, the SSU rDNA sequence alone has often not enough phylogenetic information to resolve the phylogeny of fast-evolving or very divergent sequences, leading to their misclassification. To address this problem, we tried to increase the phylogenetic signal by amplifying the complete eukaryotic rDNA cluster [i.e. the SSU rDNA, the internal transcribed spacers, the 5.8S rDNA and the large-subunit (LSU) rDNA] from environmental samples, and sequencing the SSU and LSU rDNA part of the clones. Using marine planktonic samples, we showed that surveys based on either SSU or SSU + LSU rDNA retrieved comparable diversity patterns. In addition, phylogenetic trees based on the concatenated SSU + LSU rDNA sequences showed better resolution, yielding good support for major eukaryotic groups such as the Opisthokonta, Rhizaria and Excavata. Finally, highly divergent SSU rDNA sequences, whose phylogenetic position was impossible to determine with the SSU rDNA data alone, could be placed correctly with the SSU + LSU rDNA approach. These results suggest that this method can be useful, in particular for the analysis of eukaryotic microbial communities rich in phylotypes of difficult phylogenetic ascription. [source]

Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater

ENVIRONMENTAL MICROBIOLOGY, Issue 8 2007
Xiaozhen Mou
Summary To characterize bacterioplankton functional assemblages that transform specific components of the coastal seawater dissolved organic carbon (DOC) pool, bromodeoxyuridine (BrdU) was used to label the bacterioplankton cells that were active following addition of single-DOC model compounds: two organic osmolytes [dimethylsulfoniopropionate (DMSP) and glycine betaine (GlyB)] and two aromatic monomers [para -hydroxybenzoic acid (pHBA) and vanillic acid (VanA)]. Bacterial populations were analysed based on in situ fluorescent immunodetection of BrdU incorporation followed by fluorescence-activated cell sorting (FACS). Sorted cells were then characterized by 16S rDNA-based analysis. Populations with high BrdU incorporation level (HI) developed within 8 h of introduction of 100 nM model compound. Terminal restriction fragment length polymorphisms (T-RFLP) analysis indicated that the HI populations in all four amendments were composed of bacteria from the same major taxa (phylum and subphylum levels), but the relative abundance of each differed. High-resolution clone libraries (each containing ,200 clones) showed that the HI populations in the GlyB and VanA amendments consisted of both metabolic generalists and specialists within the , -Proteobacteria (mainly members of the Roseobacter clade), , -Proteobacteria and , -Proteobacteria (mainly members of Altermonadaceae, Chromatiaceae, Oceanospirillaceae and Pseudomonadaceae). The presence of members of OM60/241, OM185, SAR11, SAR86 and SAR116 in the HI populations indicated that members of these groups can assimilate the model DOC compounds, providing some of the first glimpses into heterotrophy by members of these poorly understood environmental clusters. [source]

Bacteria associated with the rapid tissue necrosis of stony corals

ENVIRONMENTAL MICROBIOLOGY, Issue 7 2007
G. M. Luna
Summary The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio -specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for >,15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology. [source]

Prokaryotic diversity and metabolically active microbial populations in sediments from an active mud volcano in the Gulf of Mexico

ENVIRONMENTAL MICROBIOLOGY, Issue 10 2006
Robert J. Martinez
Summary In this study, ribosomes and genomic DNA were extracted from three sediment depths (0,2, 6,8 and 10,12 cm) to determine the vertical changes in the microbial community composition and identify metabolically active microbial populations in sediments obtained from an active seafloor mud volcano site in the northern Gulf of Mexico. Domain-specific Bacteria and Archaea 16S polymerase chain reaction primers were used to amplify 16S rDNA gene sequences from extracted DNA. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from each sediment depth that had been subjected to reverse transcription polymerase chain reaction amplification. Twelve different 16S clone libraries, representing the three sediment depths, were constructed and a total of 154 rDNA (DNA-derived) and 142 crDNA (RNA-derived) Bacteria clones and 134 rDNA and 146 crDNA Archaea clones obtained. Analyses of the 576 clones revealed distinct differences in the composition and patterns of metabolically active microbial phylotypes relative to sediment depth. For example, ,- Proteobacteria rDNA clones dominated the 0,2 cm clone library whereas ,-Proteobacteria dominated the 0,2 cm crDNA library suggesting , to be among the most active in situ populations detected at 0,2 cm. Some microbial lineages, although detected at a frequency as high as 9% or greater in the total DNA library (i.e. Actinobacteria, ,- Proteobacteria), were markedly absent from the RNA-derived libraries suggesting a lack of in situ activity at any depth in the mud volcano sediments. This study is one of the first to report the composition of the microbial assemblages and physiologically active members of archaeal and bacterial populations extant in a Gulf of Mexico submarine mud volcano. [source]

Quantitative, longitudinal profiling of the primate fecal microbiota reveals idiosyncratic, dynamic communities

ENVIRONMENTAL MICROBIOLOGY, Issue 3 2006
Joy Wireman
Summary We used slot blot hybridization, quantitative polymerase chain reaction (qPCR), and flow cytometry microarrays to quantify specific 16S rDNAs in weekly fecal specimens from four monkeys housed in a research vivarium for periods ranging from five to 8 months. Even in these uniformly housed and fed animals the gut microbiota is idiosyncratic, very dynamic on short timescales, and shows significant positive and negative correlations among some bacteria as well as responses to heavy metal exposure. The relative quantification (fmol targets per total fmol bacterial 16S rDNA) afforded by flow cytometry microarrays agreed well with the absolute quantification (nanogram of target DNA per nanogram of fecal DNA) afforded by slot blots and qPCR. We also noted strengths and weaknesses in inter-method comparisons for DNA-based quantification of these complex bacterial communities. [source]

Anaerobic redox cycling of iron by freshwater sediment microorganisms

ENVIRONMENTAL MICROBIOLOGY, Issue 1 2006
Karrie A. Weber
Summary The potential for microbially mediated anaerobic redox cycling of iron (Fe) was examined in a first-generation enrichment culture of freshwater wetland sediment microorganisms. Most probable number enumerations revealed the presence of significant populations of Fe(III)-reducing (approximately 108 cells ml,1) and Fe(II)-oxidizing, nitrate-reducing organisms (approximately 105 cells ml,1) in the freshwater sediment used to inoculate the enrichment cultures. Nitrate reduction commenced immediately following inoculation of acetate-containing (approximately 1 mM) medium with a small quantity (1% v/v) of wetland sediment, and resulted in the transient accumulation of NO2, and production of a mixture of gaseous end-products (N2O and N2) and NH4+. Fe(III) oxide (high surface area goethite) reduction took place after NO3, was depleted and continued until all the acetate was utilized. Addition of NO3, after Fe(III) reduction ceased resulted in the immediate oxidation of Fe(II) coupled to reduction of NO3, to NH4+. No significant NO2, accumulation was observed during nitrate-dependent Fe(II) oxidation. No Fe(II) oxidation occurred in pasteurized controls. Microbial community structure in the enrichment was monitored by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified 16S rDNA and reverse transcription polymerase chain reaction-amplified 16S rRNA, as well as by construction of 16S rDNA clone libraries for four different time points during the experiment. Strong similarities in dominant members of the microbial community were observed in the Fe(III) reduction and nitrate-dependent Fe(II) oxidation phases of the experiment, specifically the common presence of organisms closely related (, 95% sequence similarity) to the genera Geobacter and Dechloromonas. These results indicate that the wetland sediments contained organisms such as Geobacter sp. which are capable of both dissimilatory Fe(III) reduction and oxidation of Fe(II) with reduction of NO3, to NH4+. Our findings suggest that microbially catalysed nitrate-dependent Fe(II) oxidation has the potential to contribute to a dynamic anaerobic Fe redox cycle in freshwater sediments. [source]

Colonization of nascent, deep-sea hydrothermal vents by a novel Archaeal and Nanoarchaeal assemblage

ENVIRONMENTAL MICROBIOLOGY, Issue 1 2006
Elizabeth A. McCliment
Summary Active deep-sea hydrothermal vents are areas of intense mixing and severe thermal and chemical gradients, fostering a biotope rich in novel hyperthermophilic microorganisms and metabolic pathways. The goal of this study was to identify the earliest archaeal colonizers of nascent hydrothermal chimneys, organisms that may be previously uncharacterized as they are quickly replaced by a more stable climax community. During expeditions in 2001 and 2002 to the hydrothermal vents of the East Pacific Rise (EPR) (9°50,N, 104°17,W), we removed actively venting chimneys and in their place deployed mineral chambers and sampling units that promoted the growth of new, natural hydrothermal chimneys and allowed their collection within hours of formation. These samples were compared with those collected from established hydrothermal chimneys from EPR and Guaymas Basin vent sites. Using molecular and phylogenetic analysis of the 16S rDNA, we show here that at high temperatures, early colonization of a natural chimney is dominated by members of the archaeal genus Ignicoccus and its symbiont, Nanoarchaeum. We have identified 19 unique sequences closely related to the nanoarchaeal group, and five archaeal sequences that group closely with Ignicoccus. These organisms were found to colonize a natural, high temperature protochimney and vent-like mineral assemblages deployed over high temperature outflows within 92 h. When compared phylogenetically, several of these colonizing organisms form a unique clade independent of those found in mature chimneys and low-temperature mineral chamber samples. As a model ecosystem, the identification of pioneering consortia in deep-sea hydrothermal vents may help advance the understanding of how early microbial life forms gained a foothold in hydrothermal systems on early Earth and potentially on other planetary bodies. [source]

A molecular biological protocol to distinguish potentially human pathogenic Stenotrophomonas maltophilia from plant-associated Stenotrophomonas rhizophila

ENVIRONMENTAL MICROBIOLOGY, Issue 11 2005
Kathrin Ribbeck-Busch
Summary In recent years, the importance of the Gram-negative bacterium Stenotrophomonas as an opportunistic pathogen as well as in biotechnology has increased. The aim of the present study was to develop new methods for distinguishing between strains closely related to the potentially human pathogenic Stenotrophomonas maltophilia and those closely related to the plant-associated Stenotrophomonas rhizophila. To accomplish this, 58 strains were characterized by 16S rDNA sequencing and amplified ribosomal DNA restriction analysis (ARDRA), and the occurrence of specific functional genes. Based on 16S rDNA sequences, an ARDRA protocol was developed which allowed differentiation between strains of the S. maltophilia and the S. rhizophila group. As it was known that only salt-treated cells of S. rhizophila were able to synthesize the compatible solute glucosylglycerol (GG), the ggpS gene responsible for GG synthesis was used for differentiation between both species and it was confirmed that it only occurred in S. rhizophila strains. As a further genetic marker the smeD gene, which is part of the genes coding for the multidrug efflux pump SmeDEF from S. maltophilia, was used. Based on the results we propose a combination of fingerprinting techniques using the 16S rDNA and the functional genes ggpS and smeD to distinguish both Stenotrophomonas species. [source]

Molecular investigation on strain genetic relatedness and population structure of Beauveria bassiana

ENVIRONMENTAL MICROBIOLOGY, Issue 10 2003
Chengshu Wang
Summary Triplicate molecular methods, i.e. polymerase chain reaction-restriction fragment length polymorphism of the pr1 gene, microsatellite markers and 28S rDNA haplotyping by detecting the presence or absence of group I introns, were used for population study of the entomopathogenic fungus, Beauveria bassiana. The findings showed that the average genetic diversity index of geographical populations was significantly smaller than that of populations derived from insect host orders, indicating that the genetic relatedness of B. bassiana strains was highly associated with geographical locality rather than insect host species. The reproductive style of all the B. bassiana populations was found to be non-clonal. Population structure analysis revealed that the average divergent coefficient among populations of B. bassiana was far below 1 (0.1112), which indicated that there was no significant genetic differentiation between populations, and that the overall genetic diversity mainly resulted from the genetic variations within geographical populations. Statistically, genetic distances between populations were positively correlated with geographical distances, suggesting that geographical separation poses an obstacle to the possibility and frequency of genetic exchanges between populations. On the other hand, gene flow was indirectly established to occur between B. bassiana populations. [source]

The impact of grassland management on archaeal community structure in upland pasture rhizosphere soil

ENVIRONMENTAL MICROBIOLOGY, Issue 3 2003
Graeme W. Nicol
Summary The community structure of rhizosphere soil Archaea from three grassland types, associated with different management practices, was examined at a site in the Borders region of Scotland, by analysis of 16S rRNA gene fragments amplified from 16S rDNA and from rRNA. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of amplified products indicated high relative abundance within the archaeal community of two distinct lineages of non-thermophilic (group 1) Crenarchaeota. Grassland management practices influenced archaeal community structure, as characterized by both 16S rRNA- and 16S rDNA-derived DGGE profiles. One band dominated DGGE profiles in all three grassland types examined, and reproducible differences in the presence and intensity of bands were observed between profiles from managed and natural grassland sites. Analysis of 16S rRNA-derived amplicons from managed and natural grasslands at sites in the north of England and the north of Wales also indicated high relative abundance of non-thermophilic crenarchaeotes within the archaeal community. The band dominating the Scottish grassland site also dominated DGGE profiles from the English and Welsh sites, and similar differences were seen between profiles derived from soils subjected to different management regimes. The study indicates that grassland archaeal communities are dominated by Crenarchaeota, with closely related members of this lineage ubiquitous in distribution in UK upland pasture, and indicate that management practices influence the nature of the crenarchaeotal community. [source]

Phylogenetic 16S rRNA analysis reveals the presence of complex and partly unknown bacterial communities in Tito Bustillo cave, Spain, and on its Palaeolithic paintings

ENVIRONMENTAL MICROBIOLOGY, Issue 7 2002
Claudia Schabereiter-Gurtner
Summary Tito Bustillo cave (Ribadesella, Spain) contains valuable Palaeolithic paintings, which date back 15 000,20 000 years. Since 1969, the cave has been open to the public. Rock wall surfaces, spelaeothems and soils are covered by apparent biofilms of phototrophic microorganisms, which develop under artificial lighting. In addition, rock surfaces present conspicuous bacterial growth in the form of round colonies of different colours and about 1,2 mm in diameter. Even the famous Paintings Panel shows some evident microbial growth. In the present study, bacterial communities on the paintings and on the rock surfaces near the paintings were analysed by culture-independent techniques, including polymerase chain reaction (PCR) amplification of bacterial 16S rRNA genes (16S rDNA), phylogenetic sequence analyses and genetic community fingerprinting by denaturing gradient gel electrophoresis (DGGE). DGGE fingerprints showed complex bacterial community patterns. Forty-one clones matching DGGE bands of the community fingerprints were sequenced, representing about 39% of DNA fragments in the DGGE patterns. Phylogenetic sequence analyses revealed a high number of phylogenetically novel 16S rDNA sequence types and a high diversity of putatively chemotrophic and heterotrophic bacteria. Sequences were phylogenetically most closely related to the Proteobacteria (20 clones), green non-sulphur bacteria (three clones), Planctomycetales order (one clone), Cytophaga,Flexibacter, Bacteroides division (one clone) and the Actinobacteria (four clones). Furthermore, we report the presence of members of the Acidobacterium division (12 clones) in a karstic hypogean environment. Members of this phylum have not so far been detected in these particular environments. [source]

Genetic diversity of the toxic cyanobacterium Microcystis in Lake Mikata

ENVIRONMENTAL TOXICOLOGY, Issue 3 2005
Mitsuhiro Yoshida
Abstract The aim of the present study was to clarify the bloom dynamics and community composition of hepatotoxin microcystin-producing and non-microcystin-producing Microcystis genotypes in the environment. In Lake Mikata (Fukui, Japan) from April 2003 to January 2004, seasonal variation in the number of cells with microcystin (mcy) genotypes and the genetic diversity of the total population were investigated using quantitative competitive PCR and a 16S rDNA clone library, respectively. Using competitive PCR, cells with mcyA genotypes were quantified in August and October, and the ratio of the number of these mcyA genotypes to colony-forming Microcystis cells was 0.37 and 2.37, respectively. The 16S rDNA clones obtained could be divided into 12 ribotypes: a,l. Sixty-one Microcystis strains isolated from Lake Mikata during the sampling period were subjected to toxicity tests using HPLC and ELISA, PCR-based detection of the mcyA gene, and sequence analysis of the 16S rDNA. All isolates could be differentiated into 11 ribotypes (a, b, d, f, h, i, and m,q). Ribotypes b, f, i, m, n, and p had at least one strain that was a microcystin producer. In natural communities ribotypes b and f accounted for 85% of the 16S rDNA clones in August, and ribotypes b and i accounted for 24% of the clones in October. Thus, in some bloom stages the presence of microcystin genotypes identified using the 16S rDNA clone library correlated with that of mcy genotypes determined using competitive PCR. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 229,234, 2005. [source]

UNCOORDINATED PHYLOGEOGRAPHY OF BORRELIA BURGDORFERI AND ITS TICK VECTOR, IXODES SCAPULARIS

EVOLUTION, Issue 9 2010
Parris T. Humphrey
Vector-borne microbes necessarily co-occur with their hosts and vectors, but the degree to which they share common evolutionary or biogeographic histories remains unexplored. We examine the congruity of the evolutionary and biogeographic histories of the bacterium and vector of the Lyme disease system, the most prevalent vector-borne disease in North America. In the eastern and midwestern US, Ixodes scapularis ticks are the primary vectors of Borrelia burgdorferi, the bacterium that causes Lyme disease. Our phylogeographic and demographic analyses of the 16S mitochondrial rDNA suggest that northern I. scapularis populations originated from very few migrants from the southeastern US that expanded rapidly in the Northeast and subsequently in the Midwest after the recession of the Pleistocene ice sheets. Despite this historical gene flow, current tick migration is restricted even between proximal sites within regions. In contrast, B. burgdorferi suffers no barriers to gene flow within the northeastern and midwestern regions but shows clear interregional migration barriers. Despite the intimate association of B. burgdorferi and I. scapularis, the population structure, evolutionary history, and historical biogeography of the pathogen are all contrary to its arthropod vector. In the case of Lyme disease, movements of infected vertebrate hosts may play a larger role in the contemporary expansion and homogenization of the pathogen than the movement of tick vectors whose populations continue to bear the historical signature of climate-induced range shifts. [source]

Microbial community dynamics in nutrient-pulsed chemostats

FEMS MICROBIOLOGY ECOLOGY, Issue 1 2006
Militza Carrero-Colón
Abstract In nature, microbes are subject to nutrient fluxes. As the periodicity of nutrient flux lengthens, different physiological traits may be selected. The competitive exclusion principle stipulates that one organism will dominate these systems; however, interspecies interactions may produce a dynamic microbial community. These issues were investigated in chemostats pulsed with gelatin. Chemostats were run over 30 days with substrate addition continuously or at intervals of 0.5, 1 or 3 days. Growth rates were similar between pulse intervals. Ectoaminopeptidase activity levels remained relatively constant within a pulse interval. Bacterial community structure was monitored using denaturing gradient gel electrophoresis of PCR products of the 16S rRNA gene. There were dynamic changes at all periodicities; however, the pace of these changes decreased over time. Final communities were not identical between different treatments. The structure of persistent vs. active microbial populations was compared by denaturing gradient gel electrophoresis of the PCR and reverse transcriptase-PCR amplicons of 16S rDNA and rRNA templates, respectively. For all the chemostats, the rRNA profiles were not identical to the rDNA profiles for a sample. These experiments demonstrate that complex community dynamics can occur under environmental heterogeneities that are modest relative to those found in natural aquatic habitats. Furthermore, the physiological functionality of these dynamic communities was stable. [source]

Structure and diversity of Gram-negative sulfate-reducing bacteria on rice roots

FEMS MICROBIOLOGY ECOLOGY, Issue 2-3 2001
Daniel Scheid
Abstract Specific PCR assays were used to amplify the 16S rRNA genes of the Desulfobacteriaceae and the Desulfovibrionaceae from extracted environmental DNA from rice roots. 16S rDNA-based community patterns of the Desulfobacteriaceae were generated via terminal restriction fragment length polymorphism analysis from rice roots and compared with bulk soil. The molecular fingerprints showed no significant difference between rice roots and bulk soil, but changes during the vegetation period. 16S rDNA clone libraries and sequencing showed that the predominant terminal restriction fragments represented distinct phylogenetic groups. The 16S rDNA clone sequences of the Desulfobacteriaceae fell in the phylogenetic radiation of Desulfonema and Desulfosarcina or grouped within the Desulforhabdus,Syntrophobacter assemblage. Three of the latter sequences were closely affiliated with the MPN isolate EZ-2C2 from rice roots. All Desulfovibrionaceae 16S rDNA clone sequences, with one exception, were affiliated with the MPN isolate F1-7b from rice roots. The clustering of the clone sequences and the close phylogenetic affiliation with isolates from MPN enrichments from the same habitat in two cases indicated that these sequence clusters may represent predominant Gram-negative sulfate reducers on rice roots. Quantification of the bacterial abundances was accomplished by rRNA dot blot hybridization. In total the Gram-negative sulfate reducers accounted for approximately 2,3% of the total rRNA content. The relative rRNA abundance of the Desulfobacteriaceae was, at 1.4%, higher than that of the Desulfovibrionaceae (0.5%). [source]

Potentiality of the cox1 gene in the taxonomic resolution of soil fungi

FEMS MICROBIOLOGY LETTERS, Issue 1 2010
Claire Molitor
Abstract We explored the potential of the cox1 gene in the species resolution of soil fungi and compared it with the nuclear internal transcribed spacer (ITS) and small subunit (SSU)-rDNA. Conserved primers allowing the amplification of the fungal cox1 gene were designed, and a total of 47 isolates of Zygomycota and Ascomycota were investigated. The analysis revealed a lack of introns in >90% of the isolates. Comparison of the species of each of the six studied genera showed high interspecific sequence polymorphisms. Indeed, the average of nucleotide variations (4.2,11%) according to the genus, due mainly to the nucleotide substitutions, led to the taxonomic resolution of all the species studied regarding both ITS and SSU-rDNA, in which <88% were discriminated. The phylogenetic analysis performed after alignment of the cox1 gene across distant fungal species was in accordance with the well-known taxonomic position of the species studied and no overlap was observed between intra- and interspecific variations. These results clearly demonstrated that the cox1 sequences could provide good molecular markers for the determination of the species composition of environmental samples and constitute an important advance to study soil fungal biodiversity. [source]

Botryozyma mucatilis sp. nov., an anamorphic ascomycetous yeast associated with nematodes in poplar slime flux

FEMS YEAST RESEARCH, Issue 8 2004
Julia Kerrigan
Abstract A new species of Botryozyma, Botryozyma mucatilis, was isolated from the surface of free-living nematodes, Panagrellus dubius, inhabiting slime flux from hybrid poplars, Populus deltoides×trichocarpa, in Oregon, USA. This species was discovered in relatively close proximity to the teleomorphic species Ascobotryozyma americana and Ascobotryozyma cognata, both collected from P. dubius nematodes inhabiting beetle galleries in Populus spp. and Populus and Salix spp., respectively. B. mucatilis is recognized as a distinct species based on molecular and morphological data. Sequence divergence in both the D1/D2 domain of the nuclear large-subunit rDNA and internal transcribed spacer region rDNA, low DNA reassociation values, notably different amplified fragment-length polymorphic fingerprints, and significantly longer cells all support the designation of a novel species. [source]

Rhodotorula pinicola sp. nov., a basidiomycetous yeast species isolated from xylem of pine twigs

FEMS YEAST RESEARCH, Issue 2 2002
Jian-Hua Zhao
Abstract Three pink-colored yeast strains 3-1-3, 10-3-3 and 19-3-3 were isolated from xylem of surface-sterilized twigs of Pinus tabulaeformis collected from Dongling Mountain, Beijing, in different seasons. These strains were identified as Rhodotorula minuta (Saito) F.C. Harrison by conventional taxonomic characterization. However, molecular phylogenetic analysis based on internal transcribed spacer region (including 5.8S rDNA) and large-subunit rDNA D1/D2 domain sequences indicated that they represent a novel basidiomycetous yeast species, for which Rhodotorula pinicola is proposed (type strain: AS 2.2193T=CBS 9130T). The new species was most closely related to Rhodotorula laryngis Reiersöl in the R. minuta complex. [source]

Microbial communities in roots of Pinus sylvestris seedlings with damping-off symptoms in two forest nurseries as determined by ITS1/2 rDNA sequencing

FOREST PATHOLOGY, Issue 4 2009
H. Kwa
Summary A methodological molecular procedure, which included extraction and cloning of the ITS1/2 rDNA of root-associated organisms with subsequent transformation and sequencing of representative clones, was effective for detection, discrimination and determination of the frequency of the main damping-off pathogens in roots of Pinus sylvestris seedlings growing in different forest-tree nursery soils and exhibiting different rates of disease progress. Roots exhibiting slower damping-off progression were colonized by Fusarium oxysporum, Neonectria radicicola (Ascomycota) and Pythium spp. (Oomycota), which comprised 50% of the microbial community. Roots exhibiting faster damping-off progression were dominated by Thanatephorus cucumeris (Basidiomycota), which comprised 80% of the microbial community. The microbial community was more diverse in roots with slower damping-off progression (14 species) than in roots with faster disease progression (seven species). [source]

Phylogeographic variation among isolates of the Sirococcus conigenus P group

FOREST PATHOLOGY, Issue 1 2007
H. Konrad
Summary In this study the phylogeographic variation among isolates of the Sirococcus conigenus P group and the phylogenetic relationships of S. conigenus with Sirococcus clavigignenti-juglandacearum and other species previously placed in the genus Sirococcus were investigated. A collection of 33 isolates originating from Picea, Pinus and Larix in Europe, North America and Bhutan were characterized by sequence analyses of the internal transcribed spacer (ITS) region (including ITS1, 5.8S ribosomal DNA, ITS2) of the nuclear rDNA and a portion of the , -tubulin gene. In phylogenetic analyses most isolates from pine, spruce and larch formed a distinct clade, representing the P group of S. conigenus, which was separated from the T group of this pathogen. Four isolates from Picea in Europe and Canada formed a third clade within S. conigenus and these isolates are referred to as the S group. The P group consisted of five distinct ITS haplotypes, which partly differed in their optimum growth temperature and their growth rates at 25°C on malt extract agar. Nested clade analysis resolved the five haplotypes into three distinct clades and revealed significant genetic/geographic associations for some of the haplotypes. Parsimony analysis of the small subunit (18S) ribosomal DNA sequences confirmed the phylogenetic affinities between S. conigenus and S. clavigignenti-juglandacearum. In contrast, Godronia cassandrae and Hormococcus conorum, which formerly had been placed in the genus Sirococcus, were found to be only distantly related to S. conigenus and S. clavigignenti-juglandacearum. [source]

Identification of Lophodermium seditiosum and L. pinastri in Swedish forest nurseries using species-specific PCR primers from the ribosomal ITS region

FOREST PATHOLOGY, Issue 3 2005
E. Stenström
Summary Lophodermium seditiosum is a serious needle pathogen on pine, particularly in nurseries, and there is a need to detect the pathogen during its latent phase. The internal transcribed spacer (ITS) regions of the rDNA of L. seditiosum and L. pinastri were amplified with universal primers and sequenced. Sequence comparisons of the two species allowed the design of species-specific primers for the ITS regions. The primers were between 18 and 24 bp long with a minimum of 3 bp differences between the species. These primer pairs did not give any amplification of DNA from any other of the examined fungal species or from healthy Pinus sylvestris needles. It was also possible to identify either L. seditiosum or L. pinastri in infected needles with and without signs of infection using these primer pairs. The method was found to be very useful for detection of latent infections of L. seditiosum in P. sylvestris needles in nurseries. Résumé Lophodermium seditiosum est un pathogène important des aiguilles sur pins, particulièrement en pépinières, et il serait nécessaire de détecter le pathogène dans sa phase latente. Les régions ITS de L. seditiosum et L. pinastri ont été amplifiées avec des amorces universelles et séquencées. La comparaison de la séquence des deux espèces a permis de développer des amorces spécifiques pour chaque espèce dans la région ITS. Les amorces ont une longueur de 18 à 24 paires de bases avec un minimum de 3 paires de bases de différence entre espèces. Ces amorces n'ont produit aucune amplification avec l'ADN des autres espèces de champignons testées ou les aiguilles saines de Pinus sylvestris. Il a également été possible de détecter L. seditiosum ou L. pinastri avec ces amorces dans des aiguilles infectées avec ou sans signe d'infection. Cette méthode s'avère très utile pour la détection d'infections latentes de L. seditiosum dans les aiguilles de P. sylvestris en pépinières. Zusammenfassung Lophodermium seditiosum ist ein starkes Nadelpathogen an Kiefern, speziell in Baumschulen. Für den Einsatz von Bekämpfungsmassnahmen wäre es von Vorteil, wenn man das Pathogen bereits während der Latenzperiode nachweisen könnte. Die ITS Regionen der ribosomalen DNA von L. seditiosum und L. pinastri wurden mit Standardprimern amplifiziert und sequenziert. Vergleiche der Sequenzen der beiden Arten erlaubten die Entwicklung von artspezifischen Primern für die ITS Regionen. Die Primerpaare waren zwischen 18 and 24 Basenpaaren lang und wiesen einen Unterschied von mindestens drei Nukleotiden auf. Die DNA von allen anderen untersuchten Pilzarten und von gesunden Pinus sylvestris Nadeln liessen sich mit keinem dieser Primerpaare amplifiziern. Lophodermium seditiosum und L. pinastri konnten mit den Primerpaaren in infizierten Nadeln mit und ohne Symptome direkt nachgewiesen werden. Die Methode eignete sich vorzüglich zum Nachweis von latenten Infektionen von L. seditiosum in P. sylvestris Nadeln in Baumschulen. [source]

Genetic diversity of the hyperparasite Sphaerellopsis filum on Melampsora willow rusts

FOREST PATHOLOGY, Issue 5 2004
M. Liesebach
Summary The non-specific rust hyperparasite Sphaerellopsis filum occurs naturally on Melampsora rusts of many species of the genus Salix as well as on a large range of other rust genera worldwide. To study the genetic diversity of the hyperparasitic fungus 77 S. filum isolates collected from rusts on willow clones from plantations, clone collections and natural habitats of different sites were investigated using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis of the rDNA internal transcribed spacer regions including 5.8S rDNA and sequence analysis. Additionally, strains from Melampsora poplar rusts (4) and strains of Puccinia abrupta from Parthenium hysterophorus (5) and of P. obscura from Bellis perennis (1) were used for comparisons. Results of genetic analysis demonstrated distinct variation within the S. filum isolates. Two main groups with more than 32% difference between their nucleotide sequences were distinguished, indicating two taxa within S. filum. Within the first main group three profiles (I, II, III) were detected. The differences between these profiles were about 12%. The variation within each profile was very low (less than 2%). The second main group comprised two profiles (IV, V), which differed in 12 to 16% of their nucleotide positions. The isolates of group IV possessed a higher variation (up to 5%) within the group than those of the first main group (I, II, III). Group V was only represented by a single isolate. Neither interrelations between the S. filum profiles and the Melampsora genotypes nor a spatial distribution could be detected. It is remarkable that the six strains of S. filum from Puccinia rusts belong to one subgroup. Résumé Sphaerellopsis filum est un hyperparasite non spécifique des rouilles qui se rencontre naturellement sur les Melampsora affectant le genre Salix ainsi que sur une large gamme d'autres genres de rouille dans le monde entier. Pour caractériser la diversité de ce champignon hyperparasite, 77 isolats de S. filum, récoltés à partir de lésions de rouille sur des clones de plantation, des collections de clones et dans des sites naturels, ont étéétudiés par analyse PCR-RFLP des régions ITS de l'ADNr, incluant le 5.8S rDNA, et par analyse de séquences. Des souches provenant de Melampsora des peupliers (4), de Puccinia abrupta sur Parthenium hysterophorus (5) et de P. obscura sur Bellis perennis (1) ont également été utilisées pour comparaison. L'analyse génétique démontre une variation entre isolats de S. filum. Deux groupes principaux, avec plus de 32% de différence dans leur séquence nucléotidique, se distinguent, indiquant l'existence de deux taxons au sein de S. filum. A l'intérieur du premier groupe, trois profils (I, II, III) sont mis en évidence, avec une différence d'environ 12% entre profils mais très peu de variation (moins de 2%) à l'intérieur d'un profil. Le deuxième groupe comprend deux profils (IV, V) qui différent de 12 à 16% pour leur séquence nucléotidique. Les isolats du groupe IV présentent une variation intra-groupe plus importante (jusqu'à 5%) que ceux des groupes I, II et III. Le groupe V n'est représenté que par un isolat. Aucune relation n'a pu être établié entre les profils de S. filum et les génotypes de Melampsora ou la distribution spatiale. Il est à noter que les six isolats de S. filum provenant de rouilles àPuccinia appartiennent à un seul sous-groupe. Zusammenfassung Der Hyperparasit Sphaerellopsis filum kommt natürlich auf Melampsora -Rostpilzen bei Salicaceae und auf zahlreichen anderen Rostgattungen weltweit vor. Um die genetische Vielfalt dieses hyperparasitischen Pilzes zu untersuchen, wurden 77 S. filum -Proben, die von Weidenrosten in Plantagen, Klonsammlungen und von verschiedenen natürlichen Standorten stammen, isoliert. Die 5.8S-ITS-Abschnitte der rDNA wurden mit Hilfe der PCR-RFLP untersucht und Sequenzen analysiert. Zum Vergleich wurden vier Isolate von Pappelrosten sowie fünf Isolate von Puccinia abrupta von Parthenium hysterophorus und ein Isolat von Puccinia obscura von Bellis perennis herangezogen. Die Ergebnisse der genetischen Untersuchungen zeigten eine deutliche Variation zwischen den S. filum -Isolaten. Zwei Gruppen mit über 32% Unterschied in der Nukleotid-Sequenz ließen sich unterscheiden. Dies deutet auf zwei taxonomische Einheiten von Sphaerellopsis hin. Die erste Gruppe ließ sich in drei Untergruppen (I, II, III) einteilen, deren 5.8S-ITS-Profile im Mittel 12% Unterschied aufwiesen. Die Variation innerhalb dieser drei Profile war sehr gering (,2%). Die zweite Gruppe umfasste zwei Profile (IV, V), die sich an 12 bis 16% Positionen ihrer Nukleotid-Abfolge unterschieden. Die Variation innerhalb von Profil IV war höher (bis 5%) als die der Untergruppen I - III, Profil V war nur durch ein Isolat vertreten. Eine Beziehung des S. filum -Genotyps zum Melampsora -Genotyp der Weide oder der Pappel ließ sich bei dem untersuchten Material nicht nachweisen, ebenso konnte keine geographische Differenzierung gefunden werden. Auffällig ist, dass alle sechs Puccinia -Isolate einer Untergruppe angehörten. [source]

Development of species-specific PCR primers on rDNA for the identification of European Armillaria species

FOREST PATHOLOGY, Issue 5 2003
G. Sicoli
Summary Attempts to design species-specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa. Résumé Des essais ont été réalisés pour obtenir des amorces PCR spécifiques de 6 espèces européennes d'Armillaria dans les gènes de l'ARNr. Les amorces ont été développées sur la base de la variabilité de séquence nucléotidique dans les ITS et IGS (IGS1) de l'ADN ribosomal. Quatre couples d'amorces ont permis d'obtenir des produits PCR spécifiques pour A. tabescens, A. mellea et A. ostoyae. Cependant, compte tenu des très fortes similarités de séquence entre A. borealis et A. ostoyae, et entre A. cepistipes et A. gallica, il n'a pas été obtenu d'amplification spécifique pour ces taxons. Zusammenfassung Es wird über Versuche berichtet, artspezifische Primer für sechs europäische Armillariaarten in der Region der ribosomalen RNA-Gene zu entwickeln. Als Grundlage dafür diente die Variabilität der Nukleotidsequenzen der ITS- und der IGS 1-Region der ribosomalen DNA. Vier Primerpaare ergaben spezifische PCR-Produkte für A. tabescens, A. mellea und A. ostoyae. Dagegen wurden aufgrund der grossen Ähnlichkeit der Sequenzen von A. borealis und A. ostoyae sowie von A. cepistipes und A. gallica für diese Taxa keine artspezifischen Amplifikationsprodukte erhalten. [source]

Replication fork block protein, Fob1, acts as an rDNA region specific recombinator in S. cerevisiae

GENES TO CELLS, Issue 2 2002
Katsuki Johzuka
Background: The analysis of homologous recombination in the tandemly repeating rDNA array of Saccharomyces cerevisiae should provide useful information about the stability of not only the rDNA repeat but also the abundant repeated sequences on higher eukaryotic genomes. However, the data obtained so far are not yet conclusive, due to the absence of a reliable assay for detecting products of recombination in the rDNA array. Results: We developed an assay method to detect the products of unequal sister-chromatid recombination (marker-duplication products) in yeast rDNA. This assay, together with the circular rDNA detection assay, was used for the analysis. Marker-duplication occurred throughout the rDNA cluster, preferentially between nearby repeat units. The FOB1 and RAD52 genes were required for both types of recombinant formation. FOB1 showed a gene dosage effect on not only the amounts of both recombinants, but also on the copy number of the repeat. However, unlike the RAD52 gene, the FOB1 gene was not involved in homologous recombination in a non-rDNA locus. In addition, the marker-duplication products were drastically decreased in the mre11 mutant. Conclusion: Our data demonstrate that FOB1 - and RAD52 -dependent homologous recombination cause the gain and loss of a few copies of the rDNA unit, and this must be a basic mechanism responsible for amplification and reduction of the rDNA copy number. In addition, FOB1 may also play a role in the copy number regulation of rDNA tandem repeats. [source]

Microbial diversity of a sulphide spire located in the Edmond deep-sea hydrothermal vent field on the Central Indian Ridge

GEOBIOLOGY, Issue 2 2003
Joost Hoek
ABSTRACT A culture-independent molecular phylogenetic survey was carried out for a bacterial and archaeal community of a mineralized crust coating a sulphide spire, which was collected from the Edmond vent field (23° S, 69° E, 3300 m depth) on the Central Indian Ridge. Small-subunit rRNA genes (16S rDNA) were amplified from environmental DNA by PCR utilizing Bacteria-specific, and Archaea-specific 16S rDNA primers. PCR products were cloned and 26 bacterial and nine archaeal unique sequence types (phylotypes) were identified from 150 clones analysed by restriction fragment length polymorphism, representing eight and four distinct lineages, respectively. The majority (>90%) of the bacterial phylotypes group with the ,-Proteobacteria and confirms the global prevalence of ,-Proteobacteria in deep-sea hydrothermal environments. Among the ,-Proteobacteria, >40% of the phylotypes were closely related to the recently isolated deep-sea vent thermophilic chemolithoautotrophic sulphur-reducer, Nautilia lithotrophica. A single bacterial sequence was nearly identical (99% similarity) to the thermophilic hydrogen-oxidizing Hydrogenobacter thermolithotrophum, and is the first report of Hydrogenobacter at deep-sea hydrothermal vents. A majority (97%) of the archaeal phylotypes grouped with the ,Deep-sea Hydrothermal Vent Euryarchaeotal Group', a phylogenetic lineage of uncultured Archaea that have only been reported from other deep-sea hydrothermal vents on the Mid-Atlantic Ridge, East Pacific Rise, Juan de Fuca Ridge, Isu,Ogasawara Arc, Okinawa Trough and the Manus Basin. A single sequence was closely related to the hyperthermophilic sulphur-reducing Thermococcales frequently found in diverse deep-sea vent environments. Scanning electron micrographs of the mineralized crust reveal abundant filamentous, rod and coccoidal forms encased in sulphur and sulphide mineral precipitate, suggesting that the thermophilic chemolithoautorophs and sulphide-producing heterotrophs may influence the architecture and sulphur cycling of the sulphide spire. [source]

Multiple Displacement Amplification of Isolated DNA from Human Gallstones: Molecular Identification of Helicobacter DNA by Means of 16S rDNA-Based Pyrosequencing Analysis

HELICOBACTER, Issue 6 2005
Isabelle Nilsson
ABSTRACT Background., Molecular typing of Helicobacter spp. in clinical biopsy specimens has become increasingly important. By means of nested polymerase chain reaction (PCR) amplification and Southern blot analysis of the PCR amplicons, we have shown that Helicobacter spp. DNA is present in human gallstones. In this study we have investigated the possibility of using multiple displacement amplification (MDA) of isolated gallstone DNA and pyrosequencing analysis for the molecular identification of Helicobacter spp. Materials and Methods., DNA isolated from the nucleus of 33 human gallstones and one control strain were used in a MDA assay. Subsequently, pyrosequencing analysis was performed either directly on MDA-DNA using primers flanking the Helicobacter spp. 16S rDNA variable V3 region or on PCR amplicons derived from broad-range primers flanking the 16S rDNA variable V3, V4, and V9 regions. Results., Pyrosequencing analysis of 16S rDNA derived from MDA-DNA revealed that Helicobacter spp.-like DNA was present in 25 of 33 (approximately 76%) gallstones. Using an H. pylori- specific Southern blot analysis, Helicobacter spp.-like DNA was present in 20 of 33 [approximately 61%] of the gallstones. Using MDA-DNA directly in pyrosequencing analysis, Helicobacter spp.-like DNA was present in 13 of 33 [approximately 39%] gallstones. Conclusions., We conclude that multiple displacement amplification combined with pyrosequencing enables a rapid and accurate molecular typing of Helicobacter spp. from small and precious biopsy specimens. [source]

Detection of Helicobacter ganmani -Like 16S rDNA in Pediatric Liver Tissue

HELICOBACTER, Issue 5 2004
Vasundhara Tolia
ABSTRACT Background., To determine the presence of Helicobacter species in the liver biopsy specimens from children with various chronic liver diseases as data in adult literature suggests a possible role of these bacteria in their pathogenesis. Materials and methods., Paraffin sections of 61 liver biopsies of pediatric patients with miscellaneous diseases and autopsy liver tissue from 10 control subjects with no evidence of preexisting liver disease were examined for the presence of Helicobacter species by a genus-specific seminested polymerase chain reaction (PCR) assay. PCR,products of positive samples were further characterized by denaturing gradient gel electrophoresis (DGGE) and DNA-sequence analysis. Based on those results, a seminested PCR assay for H. ganmani was developed and applied to the samples. Results., On analysis, 40/61 patient samples were positive in the genus-specific Helicobacter PCR and 4/10 from the control group. The nucleotide sequences of 16S rDNA fragments were 99,100% similar to mainly Helicobacter sp. ,liver' and H. ganmani. PCR-products similar to H. canis and H. bilis were also found. The 16S rDNAs of control specimens showed similarity to Helicobacter sp. ,liver'. In the H. ganmani -specific PCR analysis 19 patients, but none of the controls, were positive. Conclusions., Amplified Helicobacter 16S rDNAs were related to Helicobacter sp. ,liver' or H. ganmani in liver biopsy specimens of pediatric patients. The possible significance of Helicobacter species in pediatric liver diseases needs to be evaluated further in prospective studies. [source]

Ribosomal DNA sequence analysis of mucosa-associated bacteria in Crohn's disease

INFLAMMATORY BOWEL DISEASES, Issue 6 2004
Tom Prindiville MD
Abstract Background: Enteric bacteria are implicated in the pathogenesis of Crohn's disease (CD); however, no specific causative organisms have been identified. Aims: This study was undertaken to correlate disease activity with changes in intestinal biota in patients with CD. Subjects: Ribosomal DNA analysis was used to explore the composition of the intestinal biota in patients with (1) CD undergoing colonoscopy, (2) CD undergoing surgical resection, and (3) no inflammatory bowel disease. Methods: Primers targeting bacterial 16S ribosomal DNA (rDNA) were used to amplify bacterial DNA associated with active CD lesions, comparable normal tissue from patients with CD, and normal control tissue. Each amplicon was cloned. Seven hundred thirty-nine rDNA clones were sequenced from 16 biopsies from CD patients, 15 surgical samples, and 10 biopsies from normal control patients. Results: Known extracellular or intracellular pathogens were not found. No rDNA sequence, phylogenetic group, or subgroup was consistently associated with CD lesions compared with normal tissues from the same patients. Colonic biopsies from CD-afflicted patients compared with biopsies from normal control subjects had an increase in facultative bacteria; in small bowel, CD patients had an increase in the Ruminococcus gnavus subgroup with a decrease in the Clostridium leptum and Prevotella nigrescens subgroups. However, differences in small bowel may have reflected individual variation rather than disease association. Surgical samples showed differences when compared with biopsy-derived samples. Conclusions: These findings suggest that CD is not caused by invasive pathogens associated specifically with the sites of lesions but that dysbiosis exists in this condition. [source]

Ticks have R2 retrotransposons but not the consensus transposon target site of other arthropods

INSECT MOLECULAR BIOLOGY, Issue 5 2005
J. Bunikis
Abstract Some copies of the large subunit rRNA genes (LSU rDNA) of most arthropods studied to date are inactivated by R-element retrotransposons at a specific target region that is highly conserved in sequence across all kingdoms of organisms. Here we report finding R2 elements in low copy numbers in the LSU rDNA of hard and soft ticks. Although the elements were inserted at the same LSU rDNA location as in insects, there were substitutions in the consensus R2 endonuclease cleavage site in the ticks and some other parasitiform mites. The substituted region comprises a critical contact point with small subunit rRNA, but in vitro structure probing analysis revealed novel, presumably stabilizing base-pairing. [source]