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

Abstract

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.

FIG. 1

PCR experiments. PCR assays were performed to check for the presence of the CFB bacterium in T. borchii Vittad. ectomycorrhizal mycelium. (A) Agarose (1%) gel electrophoresis of PCR products amplified with the b-17BO-f and UP-Reverse primers (lanes 1 to 8) or eubacterial primers UP-Forward and UP-Reverse (lanes 9 to 15). The templates used were T. borchii mycelium strains 1BO (= ATCC 96540) (lanes 1 and 9), 17BO (lanes 2 and 10), 10RA (lanes 3 and 11), Z43 (lanes 4 and 12), B2 (lanes 5 and 13), and A1 (lanes 6 and 14) and 1BO template extracted in the laboratory of P. Bonfante, University of Turin (lanes 7 and 15); no DNA was included in lanes 8 and 16. Lane M contained a fragment size marker (1-kb DNA ladder; GIBCO/BRL). (B) Agarose (1%) gel electrophoresis of PCR products amplified with the b-17BO-f and UP-Reverse primers (lanes 1 to 10) or eubacterial primers UP-Forward and UP-Reverse (lanes 11 to 20). The control templates used were ectomycorrhizae of T. borchii on T. platyphyllos, including ectomycorrhizae of 1BO (lanes 1 and 11), 17BO (lanes 2 and 12), 10RA (lanes 3 and 13), and Z43 (lanes 4 and 14). Mycelium strain 1BO was used as a positive control (lanes 5 and 15). Bacterial strains, including P. fluorescens C5 (lanes 6 and 16) and B. subtilis C15 (lanes 7 and 17), N. crassa (lanes 8 and 18), mycelial growth medium (lanes 9 and 19), and no DNA (lanes 10 and 20) were also used. Lanes M contained a fragment size marker (1-kb DNA ladder; GIBCO/BRL).

FIG. 2

Phylogenetic tree for representative 16S rRNA gene sequence from T. borchii mycelium based on nearly complete 16S rRNA sequences. The tree was derived from the evolutionary distances shown in Table 3. The two numbers at each branch node are bootstrap values based on 200 resamplings; the first number is the distance matrix value, and the second number is the parsimony bootstrap value. The sequence of the Burkholderia endosymbiont of G. margarita, an arbuscular mycorrhizal fungus, is included for comparison. Only values greater than 75 are shown. The scale bar represents a 10% difference in nucleotide sequences, as determined by measuring the lengths of the horizontal lines connecting two species. Uncu eubact, uncultivated eubacterium.

FIG. 3

Detection of the CFB bacterium associated with T. borchii Vittad. hyphal tissue (mycelial strain 17BO). (a) Phase-contrast micrograph of T. borchii hyphal tissue homogenate. (b) Same sample after hybridization with fluorescein-labeled eubacterial probe EUB338. Fluorescent CFB cells are visible. Scale bars, 5 μm.

FIG. 4

(a) Phase-contrast micrograph of T. borchii hyphal tissue homogenate. (b) Detail of the same sample after hybridization with fluorescein-labeled eubacterial probe EUB338. (c) Detail of the same sample after hybridization with CY3-labeled probe specific for b-17BO. The panel on the lower right is an overlap of panels b and c showing the same cell hybridizing with both EUB338 and STBb-654 specific for the CFB bacterium. Scale bars, 5 μm.