THE USE OF PCR-BASED TYPING METHODS TO ASSESS THE DIVERSITY OF FRANKIA NODULE ENDOPHYTES OF THE ACTINORHIZAL SHRUB CEANOTHUS

Our understanding of the actinorhizal symbiosis, in particular of the Frankia-Ceanothus association, has been hampered by the failure to iso late infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized io amplify regions of the Frankia ge nome, allowing analysis of the microsymbiont genome without first isol ating the microbe in pure culture. Root nodules were collected from si x Ceanothus spp. common to the coastal regions of the Santa Monica Mou ntains of southern California. Individual lobes were surface-sterilize d, total DNA was extracted and amplified using prokaryotic-specific pr imers. To assess the genetic diversity of Frankia endophytes in the po pulation studied, the BOX primer was used to generate genomic fingerpr ints of prokaryotic nodule inhabitants using rep-PCR. Fingerprint patt erns fell into twelve distinct groups indicating the occurrence of gen etic diversity of Frankia in the nodules sampled. DNA extracts of indi vidual lobes that gave distinct BOX-PCR fingerprints were also amplifi ed by PCR using primers directed against conserved regions of the 16S ribosomal RNA gene. The nucleotide sequences of the PCR products were determined and aligned with the corresponding region from other taxa f or phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus-infective strains.