Heavy metals adversely influence microorganisms, affecting their growth, abundance, genetic diversity, nodulation ability and efficacy. The aim of this study was to isolate and characterize free-leaving Rhizobium from soil which were artificially polluted with Cu (100, 250, and 500 mg kg-1 soil), Zn (300, 700, and 1500 mg kg-1 soil) and Pb (50, 250, and 1000 mg kg-1 soil), but also with a mixture of all these metals, and cultivated with red clover (Trifolium pratense L.), and to compare them

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... h bacteria isolated from similar type of soil, but unpolluted. Rhizobia from soil were isolated on YMA medium with or without bromothymol blue (0.00125%) as a pH-change indicator and the morpho-physiological characteristics of the colonies were examined. The number of Rhizobium was estimated using the most probable number method. Compared to the control, a decrease of rhizobia number and an increase of the metal concentration were observed. Several decameric primers (Operon Technology type) were used and a reduced polymorphism among isolated bacteria was observed. Moreover, significant differences were observed among these strains and the collection strains used as reference. Also, when primers nodCF/nodCI for detection of nod genes were used, several amplicons were obtained, different from the results obtained with similar strains isolated from unpolluted soil. These results suggest that the survival "price" of the Rhizobium in such polluted area was the alteration of some genes, including those involved in symbiosis and, probably, in nitrogen fixation.