The use of PCR‐based typing methods to assess the diversity of Frankia nodule endophytes of the actinorhizal shrub Ceanothus

Abstract

Our understanding of the actinorhizal symbiosis, in particular of the Frankia‐Ceanothus association, has been hampered by the failure to isolate infective strains in pure culture. Recently, the polymerase chain reaction (PCR) has been utilized to amplify regions of the Frankia genome, allowing analysis of the microsymbiont genome without first isolating the microbe in pure culture. Root nodules were collected from six Ceanothus spp. common to the coastal regions of the Santa Monica Mountains of southern California. Individual lobes were surface‐sterilized, total DNA was extracted and amplified using prokaryotic‐specific primers. To assess the genetic diversity of Frankia endophytes in the population studied, the BOX primer was used to generate genomic fingerprints of prokaryotic nodule inhabitants using rep‐PCR. Fingerprint patterns fell into twelve distinct groups indicating the occurrence of genetic diversity of Frankia in the nodules sampled. DNA extracts of individual lobes that gave distinct BOX‐PCR fingerprints were also amplified 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 for phylogenetic analysis. The sequences from Ceanothus nodules share a common ancestor to that of the Elaeagnus–infective strains.