IN-SITU LOCALIZATION OF AZOSPIRILLUM-BRASILENSE IN THE RHIZOSPHERE OFWHEAT WITH FLUORESCENTLY LABELED, RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES AND SCANNING CONFOCAL LASER MICROSCOPY

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization wi th fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species -specific probe for A, brasilense, a probe hybridizing to alpha subcla ss proteobacteria, and a probe specific for the domain Bacteria to ide ntify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4',6-diamidino-2 -phenylindole (DAPI) gave sufficient fluorescence signals to be detect ed with rRNA-targeted probes. Scanning confocal laser microscopy was u sed to overcome disturbing effects arising from autofluorescence of th e object or narrow depth of focus in thick specimens. This technique a lso allowed high-resolution analysis of the Spatial distribution of ba cteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 an d Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone, C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeated ly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scann ing confocal laser microscopy provided a very suitable strategy for de tailed studies of rhizosphere microbial ecology.