<?xml version="1.0" encoding="UTF-8"?><xml><records><record><source-app name="Biblio" version="7.x">Drupal-Biblio</source-app><ref-type>17</ref-type><contributors><authors><author><style face="normal" font="default" size="100%">Martin, Frank N.</style></author><author><style face="normal" font="default" size="100%">Tooley, Paul W.</style></author><author><style face="normal" font="default" size="100%">Cheryl Blomquist</style></author></authors></contributors><titles><title><style face="normal" font="default" size="100%">Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material</style></title><secondary-title><style face="normal" font="default" size="100%">Phytopathology</style></secondary-title></titles><dates><year><style  face="normal" font="default" size="100%">2004</style></year></dates><urls><web-urls><url><style face="normal" font="default" size="100%">http://dx.doi.org/10.1094/PHYTO.2004.94.6.621</style></url></web-urls></urls><volume><style face="normal" font="default" size="100%">94</style></volume><pages><style face="normal" font="default" size="100%">621-631</style></pages><language><style face="normal" font="default" size="100%">eng</style></language><abstract><style face="normal" font="default" size="100%">&lt;p class=&quot;last&quot;&gt;Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is &lt;em&gt;Phy-tophthora ramorum&lt;/em&gt;, although species such as &lt;em&gt;P. nemorosa&lt;/em&gt; and &lt;em&gt;P. pseudo-syringae&lt;/em&gt; often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the &lt;em&gt;cox&lt;/em&gt; I and II genes for detection of &lt;em&gt;Phytophthora&lt;/em&gt; spp. in general, and &lt;em&gt;P. ramorum&lt;/em&gt;, &lt;em&gt;P. nemorosa&lt;/em&gt;, and &lt;em&gt;P. pseudosyringae&lt;/em&gt; 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 &lt;em&gt;Phytophthora&lt;/em&gt; spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the &lt;em&gt;Phytophthora&lt;/em&gt; genus-specific primer pair. Using DNA from purified cultures, the &lt;em&gt;Phytophthora&lt;/em&gt; genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 &lt;em&gt;Phytophthora&lt;/em&gt; spp. evaluated, although the efficiency of amplification was lower for &lt;em&gt;P. lateralis&lt;/em&gt; and &lt;em&gt;P. sojae&lt;/em&gt; than for the other species. The genus-specific primer pair did not amplify sequences from the 30 &lt;em&gt;Pythium&lt;/em&gt; 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 &lt;em&gt;Phytophthora&lt;/em&gt; 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 &lt;em&gt;P. ramorum&lt;/em&gt;, &lt;em&gt;P. nemorosa&lt;/em&gt;, or &lt;em&gt;P. pseudo-syringae&lt;/em&gt;. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for &lt;em&gt;P. ramorum&lt;/em&gt;, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for &lt;em&gt;P. ramorum&lt;/em&gt; 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 &lt;em&gt;Phytophthora&lt;/em&gt; genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for &lt;em&gt;P. ramorum&lt;/em&gt;, 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 &lt;em&gt;Phytophthora&lt;/em&gt; genus-specific primers suggests that they will have utility for pathogen detection in other &lt;em&gt;Phytophthora&lt;/em&gt; pathosystems.&lt;/p&gt;</style></abstract></record></records></xml>