Whole cell hybridization as a tool to study <i>Frankia</i> populations in root nodules

Abstract

Molecular methods based on DNA or rRNA hybridization are powerful tools in microbial ecology for the specific detection and enumeration of bacteria unbiased by the limitations of culturability. A promising alternative to the analysis of Frankia populations in root nodules by methods based on rRNA extraction or on DNA extraction followed by the polymerase chain reaction (PCR) is the whole cell hybridization technique. This technique includes the microscopic detection of labeled probes hybridized to specific target sequences on marker molecules such as rRNA in fixed microbial cells. The analysis of uncultured Frankia populations in root nodules can reliably be performed on a subgroup level when digoxigenin‐labeled oligonucleotide probes or in vitro transcripts directed against an actinomycetes‐specific insertion on the 23S rRNA are used. Digoxigenin‐labeled probes are more suitable for in situ detection of Frankia than fluorescent probes since the sensitivity is higher and problems arising from the autofluorescence of cells and plant material are avoided. All these strategies, however, require pretreatments to increase the permeability of vesicles, hyhae and spores.