A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nipHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.

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ABSTRACT. Resistance to the antibiotic spectinomycin has been examined as a possible marker to supplement streptomycin resistance in ecological or genetic studies with rhizobia. Single step spontaneous mutants resistant to high levels of spectinomycin were isolated from 8 effective strains representing 4 species of Rhizobium. There was no evidence of cross resistance to streptomycin, and streptomycin resistant mutants were not cross resistant to spectinomycin. Minor changes in antigenic characteristics examined by immunodiffusion in agar were detected for mutants from 2 strains but these variants were still identifiable with the parent strains. Partial or full loss of symbiotic effectiveness occurred in only c. 20% of the spectinomycin resistant mutants and the resistance marker was unchanged through one plant passage. It is concluded that spectinomycin resistant mutants properly evaluated for possible pleiolropic effects should provide a useful marker system for use alone or in combination with streptomycin resistance in Rhizobium.