DIFFERENTIATION OF RHIZOBIUM STRAINS USING THE POLYMERASE CHAIN-REACTION WITH RANDOM AND DIRECTED PRIMERS

Total genomic DNA isolated from Rhizobium spp was analysed by the poly merase chain reaction (PCR) in conjunction with either arbitrary oligo nucleotide primers or a 20-base oligonucleotide primer corresponding t o a conserved nif gene promoter region. The amplified fragment length polymorphisms (amplification profiles) generated by the 2 arbitrary pr imers (RPO4 and RPO5), effectively differentiated a diverse collection of Rhizobium meliloti, R. leguminosarum by, trifolii and R. l. by, vi ciae strains. The nif-directed primer (RPOI) was also highly discrimin atory on Rhizobium DNA and generated unique amplification profiles for each strain. For selected strains, reproducible amplification profile s were obtained using a variety of DNA sources, and these were achieva ble over a 125-fold range in template concentration. Amplification pro files generated by the primers RPO5 and RPO1 were shown to be temperat ure dependant, with the RPO1 primer capable of generating amplificatio n profiles at annealing temperatures up to 65 degrees C. Reproducible amplification profiles were generated from either purified total genom ic DNA or from template DNA present in freeze-thaw bacterial cell lysa tes. Moreover, the RPO1 primer was used to positively identify specifi c R. l. by. trifolii strains directly in crude extracts prepared from squashed clover-root nodules. The significance of these results is dis cussed in relation to the development of strategies for using PCR-base d methodologies in Rhizobium ecological studies.