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Competitive PCR for quantitation of a Cytophaga-Flexibacter-Bacteroides phylum bacterium associated with the Tuber borchii Vittad. mycelium.

ABSTRACT: An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10(6) cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.

SUBMITTER: Barbieri E

PROVIDER: S-EPMC134394 | biostudies-literature | 2002 Dec

REPOSITORIES: biostudies-literature

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Competitive PCR for quantitation of a Cytophaga-Flexibacter-Bacteroides phylum bacterium associated with the Tuber borchii Vittad. mycelium.

Barbieri Elena E Riccioni Giulia G Pisano Anna A Sisti Davide D Zeppa Sabrina S Agostini Deborah D Stocchi Vilberto V

Applied and environmental microbiology 20021201 12

An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10(6) cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a m ...[more]

PMID: 12450871

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Phylogenetic characterization and in situ detection of a Cytophaga-Flexibacter-Bacteroides phylogroup bacterium in Tuber borchii vittad. Ectomycorrhizal mycelium.

Project description:Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5' and 3' ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.

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Draft Genome Sequence of Tuber borchii Vittad., a Whitish Edible Truffle.

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ESTuber db: an online database for Tuber borchii EST sequences.

Project description:BACKGROUND: The ESTuber database (http://www.itb.cnr.it/estuber) includes 3,271 Tuber borchii expressed sequence tags (EST). The dataset consists of 2,389 sequences from an in-house prepared cDNA library from truffle vegetative hyphae, and 882 sequences downloaded from GenBank and representing four libraries from white truffle mycelia and ascocarps at different developmental stages. An automated pipeline was prepared to process EST sequences using public software integrated by in-house developed Perl scripts. Data were collected in a MySQL database, which can be queried via a php-based web interface. RESULTS: Sequences included in the ESTuber db were clustered and annotated against three databases: the GenBank nr database, the UniProtKB database and a third in-house prepared database of fungi genomic sequences. An algorithm was implemented to infer statistical classification among Gene Ontology categories from the ontology occurrences deduced from the annotation procedure against the UniProtKB database. Ontologies were also deduced from the annotation of more than 130,000 EST sequences from five filamentous fungi, for intra-species comparison purposes. Further analyses were performed on the ESTuber db dataset, including tandem repeats search and comparison of the putative protein dataset inferred from the EST sequences to the PROSITE database for protein patterns identification. All the analyses were performed both on the complete sequence dataset and on the contig consensus sequences generated by the EST assembly procedure. CONCLUSION: The resulting web site is a resource of data and links related to truffle expressed genes. The Sequence Report and Contig Report pages are the web interface core structures which, together with the Text search utility and the Blast utility, allow easy access to the data stored in the database.

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