02133nas a2200157 4500008004100000245009600041210006900137300001400206490000700220520162300227100002101850700002001871700001801891700001701909856004901926 2012 eng d00aIdentification and detection of Phytophthora: reviewing our progress, identifying our needs0 aIdentification and detection of Phytophthora reviewing our progr a1080-11030 v963 a

With the increased attention given to the genus Phytophthora in the last decade in response to the ecological and economic impact of several invasive species (such as P. ramorum, P. kernoviae, and P. alni), there has been a significant increase in the number of described species. In part, this is due to the extensive surveys in historically underexplored ecosystems (e.g., forest and stream ecosystems) undertaken to determine the spread of invasive species and the involvement of Phytophthora species in forest decline worldwide (e.g., oak decline). The past decade has seen an approximate doubling in the number of described species within the genus Phytophthora, and the number will likely continue to increase as more surveys are completed and greater attention is devoted to clarifying phylogenetic relationships and delineating boundaries in species complexes. The development of molecular resources, the availability of credible sequence databases to simplify identification of new species, and the sequencing of several genomes have provided a solid framework to gain a better understanding of the biology, diversity, and taxonomic relationships within the genus. This information is much needed considering the impact invasive or exotic Phytophthora species have had on natural ecosystems and the regulatory issues associated with their management. While this work is improving our ability to identify species based on phylogenetic grouping, it has also revealed that the genus has a much greater diversity than previously appreciated.

1 aMartin, Frank, N1 aAbad, Gloria, Z1 aBalci, Yilmaz1 aIvors, Kelly uhttp://dx.doi.org/10.1094/PDIS-12-11-1036-FE03909nas a2200145 4500008004100000245017600041210006900217300001200286490000700298520334500305100002103650700002003671700002203691856005003713 2004 eng d00aMolecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material0 aMolecular detection of Phytophthora ramorum the causal agent of  a621-6310 v943 a

Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was ≈2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.

1 aMartin, Frank, N1 aTooley, Paul, W1 aBlomquist, Cheryl uhttp://dx.doi.org/10.1094/PHYTO.2004.94.6.621