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Development of two LAMP assays for the diagnostic screening of the downy mildew pathogens Peronosclerospora philippinensis, P. sacchari and P. sorghi


<div>Exotic graminicolous downy mildews are recognized as important biosecurity threats of corn, sorghum, and sugarcane in the U.S. and other parts of the world. In particular, <em>Peronosclerospora philippinensis</em> and <em>P. sacchari</em> cause destructive diseases of corn in tropical Asia and are currently listed as two out of seven USDA Select Agents for plants in the U.S. To date, <em>P. sorghi</em> is the only species in the genus <em>Peronosclerospora</em> that has been reported in the U.S. In this study, we developed two Loop-mediated isothermal amplification (LAMP) assays targeting a variable region of the mitochondrial cytochrome oxidase 2-1 spacer for the detection of the U.S. resident species <em>P. sorghi</em> (Assay 1) and exotic species <em>P. philippinensis </em>and <em>P. sacchari</em> (Assay 2). The assays were optimized for reaction time and temperature, and evaluated against other downy mildew pathogens. The LAMP reactions were optimal at 67°C (Assay 1) and 68°C (Assay 2) and a 30 minute reaction time. Optimized assays are able to detect positive samples in less than 15 minutes and for target concentrations as low as 0.5 pg/µl. No cross-reaction was observed against other <em>Peronosclerospora</em> species and other closely related pathogens downy mildew pathogens in the genera <em>Bremia</em>, <em>Baobaposis</em>, <em>Sclerophthora</em> and<em> Sclerospora</em>. These two LAMP assays are the first rapid screening methods for graminicolous downy mildew pathogens, thus enhancing our ability to protect the production of sorghum, sugarcane and corn.</div>