Frank N. Martin1, P.W. Tooley2, and R. Frederick2
The genus Phytophthora contains approximately 67 described species, many of which cause economically important plant diseases. Accurate diagnosis of pathogens to a species level has traditionally required isolation from infected tissue and classification based on morphological criteria. This can be a time intensive endeavor (for isolation and waiting for cultures to grow enough for morphological assessments to be made) that can be further complicated by morphological variation among isolates of a species, thereby requiring a certain level of taxonomic expertise with the genus by the diagnostician. The use of molecular markers can simplify and improve the accuracy of the diagnostic assay.
PCR markers based on the internal spacer sequences of the ribosomal DNA for identification of P. ramorum have been described (Garbelotto et al., 2002). An advantage to using this region for primer construction is that it is present in high copy number (thereby increasing the sensitivity of detection), however a challenge in using this region is that the rate of evolutionary change may be low, making it difficult to discriminate among more closely related species. Another genomic region that holds promise for construction of molecular markers for Phytophthora spp. is the mitochondrial DNA. Previous research has shown that specific regions are highly conserved within a species, yet divergent among even closely related species. This genome also is present in high copy number, which increases the sensitivity of the marker system.
A molecular marker system has been designed that can be used in multiplex amplifications to determine if the sample is infected by P. ramorum, other Phytophthora spp., or to determine if the extracted DNA contains PCR inhibitors. Several sets of PCR primers have been constructed that are highly specific for P. ramorum. Nested amplification can be done with these primers, thereby enabling amplification of target sequences present in low concentrations. A high degree of species specificity has been observed. When tested against 26 other Phytophthora spp., one primer pair amplified a smaller band from one other species (P. heveae, the design of this primer pair is being reevaluated) while a primer internal to these amplified only P. ramorum without background amplification from any other species.The primers amplify the fragment diagnostic for P. ramorum from all P. ramorum isolates evaluated, including those collected from California, Germany, and the Netherlands. Phytophthora genus-specific PCR primers that are compatible with the P. ramorum specific primers in multiplex PCR reactions are being tested. These will provide an additional diagnostic tool for determining if any other Phytophthora spp. are present in the plant material when the P. ramorum specific band is not amplified. Plant specific PCR primers have been developed that are compatible with the P. ramorum specific primers in multiplex PCR reactions. These will be used as internal controls to verify that the DNA extraction did not contain PCR inhibitors and reduce the risk of false negatives. The development of a real-time PCR diagnostic system is in progress.
Mitochondrial gene sequences have been used to estimate the phylogenetic relationship of P. ramorum with other species in the genus; based on cox II sequences P. ramorum was most closely related to P. hibernalis.
