The structures of bacterial communities were studied in activated sludge samples obtained from the aerobic and anaerobic zones of a wastewater treatment plant showing enhanced phosphorous removal. Samples were analyzed by in situ hybridization with oligonucleotide probes complementary to selected regions of the 16S and 23S ribosomal RNA (rRNA) characteristic for defined phylogenetic entities (genera and larger groups). The microbial community structures revealed by molecular techniques were compared with the compositions of culturable bacterial communities, obtained from the characterization of 255 isolates from tryptone-soy (TS) agar and R2A agar. These isolates were characterized by 89 physiological tests and their cellular fatty acid patterns, and identified. Culture-dependent techniques indicated the following distribution: different Aeromonas spp. (2.7-8.3% on R2A agar; 45.0-63.7% on TS agar), Acinetobacter spp. (5.4-9.0% on R2A agar; 5.0-9.1% on TS agar), Pseudomonas spp. (up to 10% on R2A agar) and Shewanella putrefaciens (up to 3.0% on R2A agar), all members of the gamma subclass of Proteobacteria, were isolated most frequently. The relatively rare isolates of the beta subclass were identified as Acidovorax spp., Alcaligenes spp., and Comamonas spp.. The Gram-positive bacteria (high DNA G+C) were assigned mainly to Arthrobacter spp., Microbacterium spp., and Mycobacterium phlei. In order to assess the in situ abundance of the most frequently isolated genus, Aeromonas, two rRNA-targeted oligonucleotide probes were developed. The two

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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 CytophagaFlexibacter-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.The mycorrhizal ascomycetous fungi belonging to the genus Tuber, commonly called truffles, are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms (23,39,45). Ectomycorrhizal fungi are present in natural and agricultural ecosystems, provide health benefits to plants, and contribute to soil nutrient cycling. The symbiotic development of mycorrhizal fungi on plant roots has been reported to be influenced by bacteria present in the mycorrhizosphere (10,16,35,47). Although various bacterial populations, such as fluorescent Pseudomonas strains and the spore-forming bacteria Micrococcus spp., Moraxella spp., Corynebacterium spp., and Staphylococcus spp., have been isolated from truffles (3,14,20), no molecular evidence has been found concerning the relationship between these organisms and their specific locations in the Tuber host tissue. There is also no information concerning the endo-and epiphytic bacteria which throughout life or during part of the life cycle may invade the tissues of the living fungus and cause unapparent and asymptomatic infections throughout the fungal life cycle and thus during production of the mycelium, contact with the host root, and development of the ectomycorrhizae and fruit bodies.Since at least 95% of all soil bacteria have not been cultured (2, 25, 36), bacteria isolated from truffles could represent only a fraction of the entire natural bacterial community associated with truffles. For this rea...

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