Phenotypic and Genetic Diversity of Rhizobia Isolated from Nodules of Clover Grown in a Zinc and Cadmium Contaminated Soil

Metal contamination can affect the diversity of microbes living in soil. We compared phenotypic and genetic characteristics of Rhizobium leguminosarum bv trifolii isolated from clovers (Trifolium pratense) found at a metal contaminated and a control site, or isolated from nodules of clovers used in most probable number (MPN) determinations of rhizobia in the metal contaminated soil. Analysis of variations in genetic patterns using BOX-PCR was used for genetic characterization. Zinc and Cd tolerance of each isolate was also determined. Rhizobia isolated from the control soil compared with the metal contaminated soil differed both genetically and phenotypically. Genetic diversity, measured by AMOVA, was high and not related to the presence or the absence of high metal concentrations. Isolates originating from the metal contaminated soil were more tolerant to both Cd (minimal inhibitory concentration [MIC 50 ] = 2.5 μM) and Zn (MIC 50 = 92.9 μM) compared with those observed for isolates of control soil (Cd MIC 50 = 2.0 μM and Zn MIC 50 = 41.9 μM). Rhizobia originating from the metal contaminated soil expressed a higher number of metal phenotypes (9) compared with isolates of control soils (6). Slow rates of metal accumulation over the years favored an adaptation of the rhizobia to the metal rather than elimination of metal sensitive organisms and the selection of a few preexisting metal tolerant organisms.