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Specific detection of quarantine species, Phytophthora ramorum, P. kernoviae and P. lateralis by loop-mediated isothermal amplification (LAMP) assay

Ayaka Hieno: River Basin Research Center, Gifu University

<div><em>Phytophthora</em> species cause destructive diseases in agriculture and forest worldwide. <em>P. ramorum</em> is a causal agent of sudden oak death, most commonly observed on <em>Camellia</em>, <em>Magnolia</em>, <em>Pieris</em> and <em>Quercus</em> spp., and has now reached epidemic proportions in North America and Europe. <em>P. kernoviae </em>causes bleeding stem cankers on members of the <em>Fagaceae</em> and foliar blight and shoot dieback on other hosts in UK and New Zealand. <em>P. lateralis</em> is highly aggressive to <em>Chamaecyparis lawsoniana</em>, a native forest tree in California and Oregon. Spread of these pathogens were probably associated with import/export ornamental plants. Therefore, an effective quarantine control of these <em>Phytophthora</em> species is highly required. In this study, we developed a species specific detection method using LAMP and simple DNA extraction kit (whole procedure completed within 30 min). LAMP primer sets were designed in rDNA-ITS, <em>cox</em>1 and <em>Ypt</em>1, and the specificity were checked with extracted DNA from 27 species of <em>Phytophthora</em> belonging to Clade 1-10, 13 species of close genus <em>Pythium</em> and <em>Phytophthium</em>, and 7 genus of other soil-borne pathogens. Selected LAMP primer sets, designed in <em>cox</em>1 gene for <em>P. ramorum</em> and <em>P. lateralis</em>, and in rDNA-ITS for <em>P. kernoviae</em> were able to detect each target from DNA samples extracted from inoculated leaves of <em>Rhododendron</em>,<em> Pieris</em> and <em>Camellia</em> within 20 min. This method can be utilized for effective detection of the three species in quarantine.</div>