Rhizobium leguminosarum bv. viciae populations in soils with increasing heavy metal contamination: abundance, plasmid profiles, diversity and metal tolerance

Populations of Rhizobium leguminosarum bv. viciae were investigated from plots of a long-term sewage sludge experiment in Braunschweig, Germany, which represented a gradient of increasing metal contamination. The number of R. leguminosarum bv. viciae decreased from 10(5) cells g(-1) soil in uncontaminated plots to between 7 and 10(2) cells g(-1) soil with increasing Zn concentration (from 50 to 400 mg kg(-1)). Rhizobia were isolated from nodules of Vicia hirsuta inoculated with dilutions of soil from seven of the plots (approximately 50 isolates per plot). The rhizobial isolates had between three and nine plasmids which varied in size from approximately 100 to 850 kb. Although a total of 49 plasmid profile groups were identified, PCR-RFLP analysis using primers which amplified an intergenic spacer (ITS) region between the 16S and 23S rRNA genes revealed only 20 groups. Ten ITS groups were found among the isolates from the uncontaminated control plot but only two groups were found in the most contaminated plot, and six to eight groups in the plots with intermediate metal contamination. Numbers of plasmid profile groups increased with moderate metal-contamination but were strongly reduced when total Zn concentrations exceeded 300 mg kg(-1). Isolates from the less contaminated plots had only three to five plasmids whereas isolates with seven to nine plasmids were abundant in the plots with metal concentrations of 200 mg kg(-1) or more. Whereas plasmid profiles indicated considerable changes in strains with increasing metal contamination, one ITS group (group 1) was present in all plots. Isolates from ITS group 1 contained only three to four plasmids in the control plots but those from the most contaminated plots had eight to nine plasmids. There was a marked increase in metal tolerance of isolates belonging to ITS group 1 as metal contamination increased, which was associated with the increase in the number of plasmids carried. However, another ITS group (group 2), which had only three large plasmids was present only in the most contaminated plot. Isolates from this group had less tolerance to Zn than many isolates from the uncontaminated plots. Possible mechanisms for the survival of these isolates in the metal contaminated soils are discussed.