Culture-independent detection of changes in root-associated bacterial populations of common bean (Phaseolus vulgaris L.) following nitrogen depletion

The structure of root-associated bacterial populations in the legume common bean (Phaseolus vulgaris L.), was studied in plants grown under nitrogen s ufficiency and under conditions inducing nitrogen deficiency. Similar cell numbers were obtained in the rhizosphere of nitrogen-amended plants as comp ared to nitrogen-deficient plants and between various root parts-tip, elong ation and branching zones-using DAPI staining. In contrast, a higher propor tion of DAPI-stained cells from the nitrogen-amended plants hybridized with a fluorescence-labeled EUB338 probe for the Bacteria domain than cells ori ginating from nitrogen-deficient plants. Shifts in the percentages of EUB33 8-reactive cells-as well as in absolute cell number-hybridizing to fluoresc ent rRNA-directed probes specific for the alpha and gamma Proteobacteria an d for high GC content gram-positive bacteria in separated root segments wer e detected between the treatments. No such differences were found using bet a and delta Proteobacteria or rRNA group I pseudomonad targeted probes. Den aturating gradient gel electrophoresis profiles of PCR products obtained fr om the same samples and amplified with Bacteria-domain targeted primers sup ported the results obtained with the whole cell hybridizations. The advanta ges and drawbacks of the techniques applied are discussed.