Differentiation of Rhizobium strains using the polymerase chain reaction with random and directed primers

Abstract

Total genomic DNA isolated from Rhizobium spp was analysed by the polymerase chain reaction (PCR) in conjunction with either arbitrary oligonucleotide primers or a 20-base oligonucleotide primer corresponding to a conserved nif gene promoter region. The amplified fragment length polymorphisms (amplification profiles) generated by the 2 arbitrary primers (RPO4 and RPO5), effectively differentiated a diverse collection of Rhizobium meliloti, R. leguminosarum bv. trifolii and R. l. bv. viciae strains. The nif-directed primer (RPO1) was also highly discriminatory on Rhizobium DNA and generated unique amplification profiles for each strain. For selected strains, reproducible amplification profiles were obtained using a variety of DNA sources, and these were achievable over a 125-fold range in template concentration. Amplification profiles generated by the primers RPO5 and RPO1 were shown to be temperature dependant, with the RPO1 primer capable of generating amplification profiles at annealing temperatures up to 65°C. Reproducible amplification profiles were generated from either purified total genomic DNA or from template DNA present in freeze-thaw bacterial cell lysates. Moreover, the RPO1 primer was used to positively identify specific R. l. bv. trifolii strains directly in crude extracts prepared from squashed clover-root nodules. The significance of these results is discussed in relation to the development of strategies for using PCR-based methodologies in Rhizobium ecological studies.