IMPROVED IN-SITU TRACKING OF RHIZOSPHERE BACTERIA USING DUAL STAININGWITH FLUORESCENCE-LABELED ANTIBODIES AND RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDES

Fluorescence-labeled antibodies and oligonucleotides were used simulta neously for the in situ identification of bacteria in mixed cultures, as well as in the rhizosphere of inoculated plants. Counterstaining wa s performed with 4'-6-diamidino-2-phenylindole (DAPI), and scanning co nfocal laser microscopy or epifluoresence microscopy with a charge-cou pled device (CCD) camera were used for detection of individual cells. This strategy gave insight into the relative abundance of an inoculate d strain, enabled the exact localization of single cells, and allowed the estimation of the metabolic activity of the bacteria in a complex specimen. Using a strain-specific monoclonal antibody for Azospirillum brasilense Wa3, we could identify this particular strain in root samp les of inoculated wheat plantlets. Strain Wa3, as well as other bacter ia colonizing the rhizosphere, were stained simultaneously with rRNA-t argeted, fluorescence-labeled oligonucleotide probes. In a co-inoculat ion experiment with A. brasilense strains Sp7 and Wa3, it was demonstr ated by in situ identification and quantitative chemoluminescence ELIS A that strain Sp7 outcompeted strain Wa3. The combined application of fluorescently labeled antibodies and oligonucleotides should be genera lly applicable for monitoring specific bacterial strains, within the b ackground of the same species, in relation to the total microbiota.