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 sufficiency and under conditions inducing nitrogen deficiency. Similar cell numbers were obtained in the rhizosphere of nitrogen-amended plants as compared to nitrogen-deficient plants and between various root parts - tip, elongation and branching zones - using DAPI staining. In contrast, a higher proportion of DAPI-stained cells from the nitrogen-amended plants hybridized with a fluorescence-labeled EUB338 probe for the Bacteria domain than cells originating from nitrogen-deficient plants. Shifts in the percentages of EUB338-reactive cells - as well as in absolute cell number - hybridizing to fluorescent rRNA-directed probes specific for the α and γ Proteobacteria and for high GC content gram-positive bacteria in separated root segments were detected between the treatments. No such differences were found using β and δ Proteobacteria or rRNA group I pseudomonad targeted probes. Denaturating gradient gel electrophoresis profiles of PCR products obtained from the same samples and amplified with Bacteriadomain targeted primers supported the results obtained with the whole cell hybridizations. The advantages and drawbacks of the techniques applied are discussed.