Showing papers on "Ribosomal DNA published in 2008"

The Epigenetics of rRNA Genes: From Molecular to Chromosome Biology

Abstract: In eukaryotes, the genes encoding ribosomal RNAs (rDNA) exist in two distinct epigenetic states that can be distinguished by a specific chromatin structure that is maintained throughout the cell cycle and is inherited from one cell to another. The fact that even in proliferating cells with a high demand of protein synthesis a fraction of rDNA is silenced provides a unique possibility to decipher the mechanism underlying epigenetic regulation of rDNA. This chapter summarizes our knowledge of the molecular mechanisms that establish and propagate the epigenetic state of rRNA genes, unraveling a complex interplay of DNA methyltransferases and histone-modifying enzymes that act in concert with chromatin remodeling complexes and RNA-guided mechanisms to define the transcriptional state of rDNA. We also review the critical role of the RNA polymerase I transcription factor UBF in the formation of active nucleolar organizer regions (NORs) and maintenance of the euchromatic state of rRNA genes.

Nucleolus: the fascinating nuclear body

Abstract: Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins. RNA polymerase I synthesizes the ribosomal RNAs and this activity is cell cycle regulated. The nucleolus reveals the functional organization of the nucleus in which the compartmentation of the different steps of ribosome biogenesis is observed whereas the nucleolar machineries are in permanent exchange with the nucleoplasm and other nuclear bodies. After mitosis, nucleolar assembly is a time and space regulated process controlled by the cell cycle. In addition, by generating a large volume in the nucleus with apparently no RNA polymerase II activity, the nucleolus creates a domain of retention/sequestration of molecules normally active outside the nucleolus. Viruses interact with the nucleolus and recruit nucleolar proteins to facilitate virus replication. The nucleolus is also a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm by nucleolus disruption. The nucleolus plays several crucial functions in the nucleus: in addition to its function as ribosome factory of the cells it is a multifunctional nuclear domain, and nucleolar activity is linked with several pathologies. Perspectives on the evolution of this research area are proposed.

Endophytic Bacterial Diversity in Rice (Oryza sativa L.) Roots Estimated by 16S rDNA Sequence Analysis

Abstract: The endophytic bacterial diversity in the roots of rice (Oryza sativa L.) growing in the agricultural experimental station in Hebei Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of rice, a pair of bacterial PCR primers (799f–1492r) was selected to specifically amplify bacterial 16S rDNA sequences directly from rice root tissues. Among 192 positive clones in the 16S rDNA library of endophytes, 52 OTUs (Operational Taxonomic Units) were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Cytophaga/Flexibacter/Bacteroides (CFB) phylum, low G+C gram-positive bacteria, Deinococcus-Thermus, Acidobacteria, and archaea. The dominant group was Betaproteobacteria (27.08% of the total clones), and the most dominant genus was Stenotrophomonas. More than 14.58% of the total clones showed high similarity to uncultured bacteria, suggesting that nonculturable bacteria were detected in rice endophytic bacterial community. To our knowledge, this is the first report that archaea has been identified as endophytes associated with rice by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in rice roots.

Genomic architecture and inheritance of human ribosomal RNA gene clusters.

Abstract: The finishing of the Human Genome Project largely completed the detailing of human euchromatic sequences; however, the most highly repetitive regions of the genome still could not be assembled. The 12 gene clusters producing the structural RNA components of the ribosome are critically important for cellular viability, yet fall into this unassembled region of the Human Genome Project. To determine the extent of human variation in ribosomal RNA gene content (rDNA) and patterns of rDNA cluster inheritance, we have determined the physical lengths of the rDNA clusters in peripheral blood white cells of healthy human volunteers. The cluster lengths exhibit striking variability between and within human individuals, ranging from 50 kb to >6 Mb, manifest essentially complete heterozygosity, and provide each person with their own unique rDNA electrophoretic karyotype. Analysis of these rDNA fingerprints in multigenerational human families demonstrates that the rDNA clusters are subject to meiotic rearrangement at a frequency >10% per cluster, per meiosis. With this high intrinsic recombinational instability, the rDNA clusters may serve as a unique paradigm of potential human genomic plasticity.

Role for perinuclear chromosome tethering in maintenance of genome stability

Abstract: Repetitive DNA sequences, which constitute half the genome in some organisms, often undergo homologous recombination. This can instigate genomic instability resulting from a gain or loss of DNA. Assembly of DNA into silent chromatin is generally thought to serve as a mechanism ensuring repeat stability by limiting access to the recombination machinery. Consistent with this notion is the observation, in the budding yeast Saccharomyces cerevisiae, that stability of the highly repetitive ribosomal DNA (rDNA) sequences requires a Sir2-containing chromatin silencing complex that also inhibits transcription from foreign promoters and transposons inserted within the repeats by a process called rDNA silencing. Here we describe a protein network that stabilizes rDNA repeats of budding yeast by means of interactions between rDNA-associated silencing proteins and two proteins of the inner nuclear membrane (INM). Deletion of either the INM or silencing proteins reduces perinuclear rDNA positioning, disrupts the nucleolus-nucleoplasm boundary, induces the formation of recombination foci, and destabilizes the repeats. In addition, artificial targeting of rDNA repeats to the INM suppresses the instability observed in cells lacking an rDNA-associated silencing protein that is typically required for peripheral tethering of the repeats. Moreover, in contrast to Sir2 and its associated nucleolar factors, the INM proteins are not required for rDNA silencing, indicating that Sir2-dependent silencing is not sufficient to inhibit recombination within the rDNA locus. These findings demonstrate a role for INM proteins in the perinuclear localization of chromosomes and show that tethering to the nuclear periphery is required for the stability of rDNA repeats. The INM proteins studied here are conserved and have been implicated in chromosome organization in metazoans. Our results therefore reveal an ancient mechanism in which interactions between INM proteins and chromosomal proteins ensure genome stability.

A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics.

Abstract: Streptomycetes are a complex group of actinomycetes that produce diverse bioactive metabolites of commercial significance. Systematics can provide a useful framework for identifying species that may produce novel metabolites. However, previously proposed approaches to the systematics of Streptomyces have suffered from either poor interlaboratory comparability or insufficient resolution. In particular, the Streptomyces griseus 16S rRNA gene clade is the most challenging and least defined group within the genus Streptomyces in terms of phylogeny. Here we report the results of a multilocus sequence analysis scheme developed to address the phylogeny of this clade. Sequence fragments of six housekeeping genes, atpD, gyrB, recA, rpoB, trpB and 16S rRNA, were obtained for 53 reference strains that represent 45 valid species and subspecies. Analysis of each individual locus confirmed the suitability of loci and the congruence of single-gene trees for concatenation. Concatenated trees of three, four, five and all six genes were constructed, and the stability of the topology and discriminatory power of each tree were analysed. It can be concluded from the results that phylogenetic analysis based on multilocus sequences is more accurate and robust for species delineation within Streptomyces. A multilocus phylogeny of six genes proved to be optimal for elucidating the interspecies relationships within the S. griseus 16S rRNA gene clade. Our multilocus sequence analysis scheme provides a valuable tool that can be applied to other Streptomyces clades for refining the systematic framework of this genus.

Analysis of fungal flora in indoor dust by ribosomal DNA sequence analysis, quantitative PCR, and culture.

Abstract: In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed.

Quantification of Diatom and Dinoflagellate Biomasses in Coastal Marine Seawater Samples by Real-Time PCR

Abstract: Two real-time PCR assays targeting the small-subunit (SSU) ribosomal DNA (rDNA) were designed to assess the proportional biomass of diatoms and dinoflagellates in marine coastal water. The reverse primer for the diatom assay was designed to be class specific, and the dinoflagellate-specific reverse primer was obtained from the literature. For both targets, we used universal eukaryotic SSU rDNA forward primers. Specificity was confirmed by using a BLAST search and by amplification of cultures of various phytoplankton taxa. Reaction conditions were optimized for each primer set with linearized plasmids from cloned SSU rDNA fragments. The number of SSU rDNA copies per cell was estimated for six species of diatoms and nine species of dinoflagellates; these were significantly correlated to the biovolumes of the cells. Nineteen field samples were collected along the Swedish west coast and subjected to the two real-time PCR assays. The linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. For diatoms, linear regression of the number of SSU rDNA copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates. The method described will be useful for estimating the proportion of dinoflagellate versus diatom biovolume or biomass and the absolute diatom biovolume or biomass in various aquatic disciplines.

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Abstract: Synthesis of ribosomal RNAs (rRNAs) is the major transcriptional event in proliferating cells. In eukaryotes, ribosomal DNA (rDNA) is transcribed by RNA polymerase I from a multicopy locus coexisting in at least two different chromatin states. This heterogeneity of rDNA chromatin has been an obstacle to defining its molecular composition. We developed an approach to analyze differential protein association with each of the two rDNA chromatin states in vivo in the yeast Saccharomyces cerevisiae. We demonstrate that actively transcribed rRNA genes are largely devoid of histone molecules, but instead associate with the high-mobility group protein Hmo1.

Phytoplasma phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of 'Candidatus Phytoplasma'.

Abstract: Phytoplasma phylogenetics has focused primarily on sequences of the non-coding 16S rRNA gene and the 16S–23S rRNA intergenic spacer region (16–23S ISR), and primers that enable amplification of these regions from all phytoplasmas by PCR are well established. In this study, primers based on the secA gene have been developed into a semi-nested PCR assay that results in a sequence of the expected size (about 480 bp) from all 34 phytoplasmas examined, including strains representative of 12 16Sr groups. Phylogenetic analysis of secA gene sequences showed similar clustering of phytoplasmas when compared with clusters resolved by similar sequence analyses of a 16–23S ISR–23S rRNA gene contig or of the 16S rRNA gene alone. The main differences between trees were in the branch lengths, which were elongated in the 16–23S ISR–23S rRNA gene tree when compared with the 16S rRNA gene tree and elongated still further in the secA gene tree, despite this being a shorter sequence. The improved resolution in the secA gene-derived phylogenetic tree resulted in the 16SrII group splitting into two distinct clusters, while phytoplasmas associated with coconut lethal yellowing-type diseases split into three distinct groups, thereby supporting past proposals that they represent different candidate species within ‘Candidatus Phytoplasma’. The ability to differentiate 16Sr groups and subgroups by virtual RFLP analysis of secA gene sequences suggests that this gene may provide an informative alternative molecular marker for pathogen identification and diagnosis of phytoplasma diseases.

A ribosomal DNA-based framework for the detection and quantification of stress-sensitive nematode families in terrestrial habitats.

Abstract: Indigenous communities of soil-resident nematodes have a high potential for soil health assessment as nematodes are diverse, abundant, trophically heterogeneous and easily extractable from soil. The conserved morphology of nematodes is the main operational reason for their under-exploitation as soil health indicators, and a user-friendly biosensor system should preferably be based on nonmorphological traits. More than 80% of the most environmental stress-sensitive nematode families belong to the orders Mononchida and Dorylaimida. The phylogenetic resolution offered by full-length small subunit ribosomal DNA (SSU rDNA) sequences within these two orders is highly different. Notwithstanding several discrepancies between morphology and SSU rDNA-based systematics, Mononchida families (indicated here as M1?M5) are relatively well-supported and, consequently, family-specific DNA sequences signatures could be defined. Apart from Nygolaimidae and Longidoridae, the resolution among Dorylaimida families was poor. Therefore, a part of the more variable large subunit rDNA (? 1000 bp from the 5'-end) was sequenced for 72 Dorylaimida species. Sequence analysis revealed a subclade division among Dorylaimida (here defined as D1?D9, PP1?PP3) that shows only distant similarity with `classical? Dorylaimid systematics. Most subclades were trophically homogeneous, and ? in most cases ? specific morphological characteristics could be pinpointed that support the proposed division. To illustrate the practicability of the proposed molecular framework, we designed primers for the detection of individual subclades within the order Mononchida in a complex DNA background (viz. in terrestrial or freshwater nematode communities) and tested them in quantitative assays (real-time polymerase chain reaction). Our results constitute proof-of-principle for the concept of DNA sequence signatures-based monitoring of stress sensitive nematode families in environmental samples

Location and expression of ribosomal RNA genes in grasshoppers: abundance of silent and cryptic loci.

Abstract: We investigate regularities and restrictions in chromosome location of ribosomal RNA genes, analysed by fluorescent in situ hybridization (FISH), and their phenotypic expression assessed by nucleolus formation at first meiotic prophase cells, analysed by silver impregnation, in 49 grasshopper species. High variation was found for rDNA location between species within most genera analysed. The mean haploid number of rDNA loci detected by FISH was 2.47, but some species had up to 10 loci. Chromosome distribution of rDNA loci differed between the Gomphocerinae and Oedipodinae subfamilies, most loci being proximal to the centromere in the former and distal to it in the latter. Chromosomes 2, 3 and X frequently carried rDNA in Gomphocerinae species with 2n♂=17 chromosomes, whereas chromosomes 6 and 9 were the most frequent rDNA locations in the Oedipodinae. About 13% of the 126 rDNA loci detected by FISH were silent, although this figure might be even higher. The comparison of FISH and silver-impregnation results also suggested the existence of cryptic NORs, i.e. those forming small nucleoli with no apparent presence of rDNA revealed by FISH. This was especially clear after the same cells in two species were sequentially treated with both silver impregnation and FISH. The abundance of silent and cryptic loci might thus suggest that rDNA spreads through grasshopper genomes by the Dubcovsky and Dvorak mechanism—that is, the transposition of a few rRNA genes to new chromosome locations, their amplification giving rise to new NORs, and the elimination of the old NORs. The cryptic NORs might correspond to nascent NORs, i.e. a few rRNA gene copies moved to new locations, whereas the inactive rDNA loci might correspond to those being in the process of elimination.

Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre

Abstract: High-throughput identification of rRNA gene-containing clones in large insert metagenomic libraries is difficult, because of the high background of host ribosomal RNA (rRNA) and rRNA genes. To address this challenge, a membrane hybridization method was developed to identify all bacterial small subunit rRNA-containing fosmid clones of microbial community DNA from seven different depths in the North Pacific Subtropical Gyre. Out of 101,376 clones screened, 751 rDNA-containing clones were identified that grouped in approximately 60 different clades. Several rare sequences only remotely related to known groups were detected, including a Wolbachia-related sequence containing a putative intron or intervening sequence, as well as seven sequences from Order Myxococcales not previously detected in pelagic habitats. Stratified, depth-specific population structure was evident within both cultured and uncultured lineages. Conversely, some eurybathyal members of the genera Alcanivorax and Rhizobium shared identical small subunit ribosomal DNA sequences that were distributed from surface waters to the 4000 m depth. Comparison with similar analyses in Monterey Bay microbial communities revealed previously recognized, as well as some distinctive, depth-stratified partitioning that distinguished coastal from open ocean bacterioplankton populations. While some bias was evident in fosmid clone recovery in a few particular lineages, the overall phylogenetic group recovery and distributions were consistent with previous studies, as well as with direct shotgun sequence data from the same source DNA.

Haploinsufficiency of RPS14 in 5q- syndrome is associated with deregulation of ribosomal- and translation-related genes.

Abstract: We have previously demonstrated haploinsufficiency of the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region, in the 5q- syndrome. Patients with Diamond-Blackfan anaemia (DBA) show haploinsufficiency of the closely related ribosomal protein RPS19, and show a consequent downregulation of multiple ribosomal- and translation-related genes. By analogy with DBA, we have investigated the expression profiles of a large group of ribosomal- and translation-related genes in the CD34(+) cells of 15 myelodysplastic syndrome (MDS) patients with 5q- syndrome, 18 MDS patients with refractory anaemia (RA) and a normal karyotype, and 17 healthy controls. In this three-way comparison, 55 of 579 ribosomal- and translation-related probe sets were found to be significantly differentially expressed, with approximately 90% of these showing lower expression levels in the 5q- syndrome patient group. Using hierarchical clustering, patients with the 5q- syndrome could be separated both from other patients with RA and healthy controls solely on the basis of the deregulated expression of ribosomal- and translation-related genes. Patients with the 5q- syndrome have a defect in the expression of genes involved in ribosome biogenesis and in the control of translation, suggesting that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis.

Exophiala spinifera and its allies: diagnostics from morphology to DNA barcoding.

Abstract: Diagnostic features of morphology, physiology, serology and genetics of species belonging to the Exophiala spinifera clade (including 11 species: Exophiala oligosperma, E. spinifera, E. xenobiotica, E. jeanselmei, E. exophialae, E. nishimurae, E. bergeri, E. nigra, Rhinocladiella similis, Ramichloridium basitonum and Phaeoannellomyces elegans), comprising a large number of human-associated Exophiala species, are summarized. Several species have closely similar morphological characters and physiological profiles. Taxonomy is therefore primarily based on sequence diversity of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA). Multilocus sequencing has shown that ITS is reliable for identification of the species in this clade, and is a therefore a good candidate for barcoding species of Exophiala. Species-specific fragments were searched in the ITS region of species in the Exophiala spinifera clade and can be used to design probes for diagnosis by hybridization.

Identification of dermatophytes by sequence analysis of the rRNA gene internal transcribed spacer regions.

Abstract: Identification of dermatophytes using the traditional method is sometimes problematic because of atypical microscopic or macroscopic morphology. The aim of this study was to evaluate the feasibility of using sequencing of the ribosomal internal transcribed spacer (ITS)1 and ITS2 regions for identification of 17 dermatophyte species. The ITS regions of 188 strains (62 reference strains and 126 clinical isolates) were amplified by PCR and sequenced. Species identification was made by sequence comparison with an in-house database comprising ITS sequences of type or neotype strains or by BLAST searches for homologous sequences in public databases. Strains producing discrepant results between conventional methods and ITS sequence analysis were analysed further by sequencing the D1–D2 domain of the large-subunit rRNA gene for species clarification. The identification rates by ITS1 and ITS2 sequencing were higher than 97%. Based on reference sequences of type or neotype strains, it was noted that most strains of Trichophyton mentagrophytes were misidentifications of Trichophyton interdigitale. In addition, barcode sequences were present in species of the Microsporum canis complex and Trichophyton rubrum complex. These barcode sequences are useful for species delineation when the results of ITS sequencing are ambiguous. In conclusion, ITS sequencing provides a very accurate and useful method for the identification of dermatophytes.

Evidence from population genetics that the ectomycorrhizal basidiomycete Laccaria amethystina is an actual multihost symbiont.

Abstract: It is commonly assumed that ectomycorrhizal (ECM) fungi associated with temperate forest tree roots are not host-specific. Because this assumption relies on species delineations based on fruitbodies morphology or ribosomal DNA sequences, host-specific, cryptic biological species cannot be ruled out. To demonstrate that Laccaria amethystina has true generalist abilities, we sampled 510 fruitbodies on three French sites situated 150–450 km away from each other. At each site, populations from monospecific stands (Abies alba, Castanea europea and Fagus sylvatica) or mixed stands (F. sylvatica + Quercus robur or Q. robur +Carpinus betulus) were sampled. Three different sets of markers were used for genotyping: (i) five microsatellite loci plus the ribosomal DNA intergenic spacer, (ii) the mitochondrial large ribosomal DNA subunit, and (iii) direct amplification of length polymorphism (DALP), a new method for fungi providing dominant markers. Evidence for allogamous populations (with possible inbreeding at local scale) and possibly for biparental mitochondrial inheritance was found. All markers congruently demonstrated that L. amethystina populations show little structure at this geographical scale, indicating high gene flow (as many as 50% of founding spores in all populations being of external origin). Our results also showed that host species contributed even less to population differentiation, and there was no evidence for cryptic biological species. This first in situ demonstration of a true multihost ability in an ECM species is discussed in terms of ecology and evolutionary biology.

Chromosomal localization and copy number of 18S + 28S ribosomal RNA genes in evolutionarily diverse mosquitoes (Diptera, Culicidae).

Abstract: In situ hybridization using 3H-labeled 18S and 28S ribosomal DNA (rDNA) probes from Aedes albopictus was performed on the mitotic chromosomes of 20 species of mosquitoes belonging to 8 genera of subfamilies Culicinae and Anophelinae In all but one species examined, the rDNA family was localized to a single chromosome per haploid genome Aedes triseriatus was the only exception, with the rDNA cistrons present on chromosome 1 and on chromosome 3 The ribosomal RNA genes were located on chromosome 1 in Ae albopictus, Ae aegypti, Ae flavopictus, Ae seatoi, A polynesiensis, Ae alcasidi, Ae annandalei, Ae mascarensis, Ae hendersoni, Ae atropalpus, Ae epactius, Culex pipiens quinquefasciatus, Wyeomyia smithii, and Sabethes cyaneus; chromosome 2 in Ae mediovittatus and Haemagogus equinus; chromosome 3 in Armigeres subalbatus and Tripteroides bambusa; and the heteromorphic X and Y chromosomes in Anopheles quadrimaculatus The variation in the location of ribosomal RNA genes on the different chromosomes and at different positions on the chromosome arm among the mosquito species examined is suggestive of considerable chromosome repatterning through translocations and inversions in the karyotypic evolution of mosquitoes Dot-blot hybridization was used to estimate copy number of rRNA genes; the copy number per haploid genome ranged from 39 +/- 327 in Sa cyaneus to 1023 +/- 6814 in Ae flavopictus

New insights into the identity and origin of the causal agent of oak powdery mildew in Europe

Abstract: Summary Powdery mildew is the most common disease on oaks in Europe where it was first recorded at the beginning of the 20th century Yet, little is known about the origin of the causal agent In this study, we analysed the variability of the ribosomal DNA (rDNA) of the pathogen The internal transcribed spacer (ITS) region, the 58S rRNA coding gene and the intergenic spacer (IGS) of the rDNA of 33 European (mostly French) samples of oak powdery mildew were amplified using polymerase chain reaction (PCR) and PCR products were subsequently sequenced Four different haplotypes were obtained for ITS among the various samples (ITSA, ITSB, ITSC and ITSD) Each ITS sequence corresponded to a different IGS sequence The comparison of ITS sequences obtained with sequences accessible in the GenBank database revealed very high homologies with different taxa Of these, three taxa had already been described on oaks in Europe, ie Erysiphe alphitoides (100% homology with ITSA), Erysiphe hypophylla (994% homology with ITSC) and Phyllactinia guttata (9764% homology with ITSD) Our data also confirmed the 100% homology between ITSA and the sequence described for Oidium mangiferae, the agent of mango powdery mildew The fourth haplotype, ie ITSB, represented by nearly 25% samples, showed 100% homology with the recently described Erysiphe quercicola from Quercus spp in Asia, and several tropical and sub-tropical powdery mildew species, including Oidium heveae, a major pathogen of rubber trees worldwide Our results suggest that oak powdery mildew might originate from host shifts of tropical Erysiphe species introduced to Europe through infected exotic host plants

Molecular phylogenetic studies within the Xylariaceae based on ribosomal DNA sequences

Abstract: The Xylariaceae (Xylariales, Ascomycota) are considered one of the largest families of filamentous Ascomycetes. To infer the evolutionary relationships of some genera considered within the Xylariaceae, the 5.8S rRNA gene and ITS2 sequences of 100 isolates covering 15 genera and 62 taxa, were analysed phylogenetically. To obtain an accurate view on the evolutionary relationships of genera within Xylariaceae, four different sequence analysis methods (i.e. Parsimony, Neighbor-joining, Maximum-likelihood and Bayesian analyses) were employed, and a consensus phylogram was obtained to integrate data from all these mentioned approaches. Rates of congruence between topologies of the trees generated were also estimated by different methods. The phylogenetic reconstructions showed a reasonable degree of correlation between the sequence data and the proposed morphological classification schemes only for some genera within the family. The Hypoxylon-related genera included in the study (i.e. Hypoxylon, Annulohypoxylon, Biscogniauxia, Camillea, Creosphaeria, Whalleya and Daldinia) appeared closely related within a large clade in all the trees. Nemania always clusters apart from this clade of Hypoxylon-related genera, often found at the base of the tree. Phylogenetic reconstruction supported a polyphyletic origin for the genera Xylaria and Rosellinia, suggesting that these must be considered as large and complex genera, made up of a mixture of weakly related species. Kretzschmaria and Stilbohypoxylon appeared to be highly related to some Xylaria species. Finally, Entoleuca seems to be closely related to Rosellinia. In summary, this study suggests the need for further revision of the generic concepts and diagnostic characters within the Xylariaceae.

Species-level identification of Bacillus strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis

Abstract: The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.