Phylogenetic characterization and in situ detection of a Cytophaga-Flexibacter-Bacteroides phylogroup bacterium in Tuber borchii Vittad, ectomycorrhizal mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straight forward approach in which a combination of morphological and molecular meth ods was used to survey the presence of potentially endo- and epiphytic bact eria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5' and 3' ends of th e 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequenci ng, and phylogenetic analyses, The 16S rDNA was amplified directly from fou r pure cultures of T. borchii Vittad, mycelium, A nearly full-length sequen ce of the gene coding for the prokaryotic small-subunit rRNA was obtained f rom each T. borchii mycelium studied. The 16S rDNA sequences were almost id entical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroide s (CFB) phylogroup which had not been described previously. In situ detecti on of the CFB bacterium in the hyphal tissue of the fungus T. borchii was c arried out by using 16S rRNA-targeted oligonucleotide probes for the eubact erial domain and the Cytophaga-Flexibacter phylum, as well as a probe speci fically designed for the detection of this mycelium-associated bacterium. F luorescent in situ hybridization showed that all three of the probes used b ound to the mycelium tissue. This study provides the first direct visual ev idence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fu ngus of the genus Tuber.