The stability of identification markers was examined for two Rhizobium galegae inoculant strains after 5 years in the field. The two strains are genetically closely related, but differ in their lipopolysaccharides. Strain HAMBI 540 has lipopolysaccharide of the rough type, whereas that of strain HAMBI 1461 is of the smooth type. The properties that were examined for 10 field isolates of each inoculant type were symbiotic phenotype, phage type, intrinsic antibiotic resistance, maximum growth temperature, lipopolysaccharide and total soluble protein patterns, immunological properties, DNA restriction profiles, and DNA hybridization patterns, which were determined by using nifHDK and recA sequences as probes. Of these properties, all remained stable in soil, with the exception of some variation in intrinsic antibiotic resistance and the acquisition of an extra EcoRI restriction fragment by one of the isolates. Thus, both the rough and the smooth lipopolysaccharide phenotypes persisted equally well in soil. The stability of markers that are used for the identification of rhizobial field isolates is of crucial importance for successful interpretation of results from inoculation and competition experiments. Identification methods commonly used are phage typing (6), intrinsic antibiotic resistance (IAR) (12, 14, 22), protein profiles, and serological properties (3, 12, 15, 33, 36), either separately or in combination. These methods identify wild-type strains; the use of genetically marked mutant strains is not dealt with here. IAR (2, 5, 20), protein profiles (19), serology (5, 10, 11, 19, 20), phage typing (6), and plasmid profiles (5) have also been used for evaluating the diversity of indigenous rhizobia in soil. DNA restriction profiles have been used to study the genetic diversity within serogroup 123 of Bradyrhizobiulm japonicum (37, 38).
