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Applied and Environmental Microbiology, November 2000, p. 5035-5042, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

Elena Barbieri,1 Lucia Potenza,1 Ismaela Rossi,1 Davide Sisti,2 Giovanna Giomaro,2 Simona Rossetti,3 Claudia Beimfohr,4 and Vilberto Stocchi1,*

"Giorgio Fornaini" Institute of Biochemistry1 and Institute of Botany,2 University of Urbino, 61029 Urbino, and Water Research Institute, CNR, 00198 Rome,3 Italy, and Vermicon AG, 80992 Munich, Germany4

Received 29 September 1999/Accepted 2 August 2000

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.

* Corresponding author. Mailing address: "Giorgio Fornaini" Institute of Biochemistry, University of Urbino, Via Saffi, 2, 61029 Urbino (PU), Italy. Phone: 39 0722 305262. Fax: 39 0722 320188. E-mail: vstocchi{at}uniurb.it.

Applied and Environmental Microbiology, November 2000, p. 5035-5042, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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