HEAVY-METALS FROM PAST APPLICATIONS OF SEWAGE-SLUDGE DECREASE THE GENETIC DIVERSITY OF RHIZOBIUM-LEGUMINOSARUM BIOVAR TRIFOLII POPULATIONS

White clover plants grown at a site contaminated with heavy metals following applications of sewage sludge were found to have small white root nodules containing ineffective rhizobia (S isolates) which had identical plasmid profiles, unlike the diverse profiles of effective rhizobia from root nodules on adjacent control plots. Our paper supports an earlier suggestion that the ineffective S isolates of Rhizobium from nodules of white clover grown on heavy-metal contaminated soil represent a single strain. These new data include restriction fragment length polymorphism (RFLP) studies using probes specific for a chromosomally-located gene (lac), a plasmid-located symbiotic gene (nifH,D) and a repeated sequence specific for Rhizobium leguminosarum bv. trifolii (RtRS). RFLP patterns of isolates from control plots indicated that although these strains showed variation, they were related to one another but not to the S isolates. We also demonstrated that although the S isolates were ineffective on white clover, they formed normal nodules on subterranean clover, which were effective in nitrogen fixation. However, they ineffectively nodulated red clover and were unable to nodulate Vicia hirsuta. Thus the population of R. leguminosarum bv. trifolii had been radically altered by long-term exposure to heavy metal contamination, apparently losing those agronomically-important strains capable of forming effective symbiotic associations with white and red clover.