PCR-based DNA Markers for Identifying Hybrids within Phytophthora alni

Two pairs of oligonucleotide primers were designed for the polymerase chain reaction (PCR)-based detection and differential identification of naturally occurring interspecific hybrid types (subspecies) of Phytophthora alni, all of which cause collar rot of alder trees. Primer pairs were derived from randomly amplified polymorphic DNA (RAPD) fragments that were unique to various subspecies of this alder pathogen. The primer pair set, SAP1/SAP2 (SAP), was derived from a 0.93-kb RAPD fragment amplified from P. alni ssp. alni. The primer pair set, SWAP1/SWAP2 (SWAP), was derived from a 1.13-kb fragment amplified from P. alni ssp. uniformis. Patterns of SAP and SWAP amplification enabled distinction among the three subspecies. No PCR products were amplified from isolates of 31 other Phytophthora spp. examined, including P. cambivora and P. fragariae, the suspected progenitors of P. alni. The SAP and SWAP primer sets were able to detect a minimum of 10 pg of DNA from pure cultures or DNA extracted from as few as 10 zoospores. Pathogen DNA could also be amplified directly from bark lesions of artificially inoculated and naturally infected common alders and from lesions developed on common cherry-laurel leaves used in baiting the pathogen from infested soil. Direct detection of pathogen DNA from alder tissue using SAP and SWAP primer sets should prove useful in developing measures for effective quarantine and management of P. alni