Development of clade- (Roseobacter and Alteromonas) and taxon-specific oligonucleotide probes to study interactions between toxic dinoflagellates andtheir associated bacteria

Bacteria associated with toxic algae in culture have been implicated in the enhancement of algal toxin production and auto-toxigenesis. Toxigenic and non-toxigenic bacterial isolates from Alexandrium spp. have been determined to belong predominantly to the alpha- and gamma- subclasses of the class P roteobacteria. Within these subclasses, the isolates were further classifie d into two clades that were phylogenetically affiliated with (1) the genus Roseobacter. a newly identified group that is of major significance within the alpha -Proteobacteria and (2) the genus Alteromonas. Specific 16S rRNA sequence signatures were identified for these clades as well as for three p airs of isolates and for one subcluster within the Roseobacter clade and fo r three clusters representing eight isolates in the Alteromonas clade. Oligonucleotide probes complementary to these regions were designed and the ir specificities were tested using dot-blot and whole-cell in situ hybridiz ations of target and non-target strains. The empirically determined dissoci ation temperatures of the probes ranged between 55 and 65 making them appli cable as a set for screening environmental samples. In situ hybridization o f bacteria in cultures of Alexandrium spp. with the clade-specific probes s howed a loose association of these bacteria within the phycosphere. in addi tion, the fluorescent signal of the probes was bright and easily distinguis hable from autofluorescent bacteria and the dinoflagellates.