Improved in situ tracking of rhizosphere bacteria using dual staining with fluorescence-labeled antibodies and rRNA-targeted oligonucleotides

Fluorescence-labeled antibodies and oligonucleotides were used simultaneously for the in situ identification of bacteria in mixed cultures, as well as in the rhizosphere of inoculated plants. Counterstaining was performed with 4'-6-diamidino-2-phenylindole (DAPI), and scanning confocal laser microscopy or epifluoresence microscopy with a charge-coupled device (CCD) camera were used for detection of individual cells. This strategy gave insight into the relative abundance of an inoculated 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 samples of inoculated wheat plantlets. Strain Wa3, as well as other bacteria colonizing the rhizosphere, were stained simultaneously with rRNA-targeted, fluorescence-labeled oligonucleotide probes. In a co-inoculation experiment with A. brasilense strains Sp7 and Wa3, it was demonstrated by in situ identification and quantitative chemoluminescence ELISA that strain Sp7 outcompeted strain Wa3. The combined application of fluorescently labeled antibodies and oligonucleotides should be generally applicable for monitoring specific bacterial strains, within the background of the same species, in relation to the total microbiota.