Specific detection of the wheat blast pathogen (Magnaporthe oryzae Triticum) by loop-mediated isothermal amplification
Jarred Yasuhara-Bell: Kansas State University
<div>Wheat blast, caused by <em>Magnaporthe oryzae </em>Triticum (MoT) pathotype, is an economically important fungal disease of wheat. Fusarium head scab of wheat produces symptoms similar to blast and can cause confusion in the field. Currently, no in-field diagnostic exists for MoT. To address this, loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown specific for <em>M. oryzae </em>and MoT, respectively. Assay specificity was determined using gDNA from 104 <em>M. oryzae </em>strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included <em>Fusarium graminearum </em>DNA. Sensitivity was assessed using 10-fold serial dilutions of MoT gDNA. PoT2- and MoT3-based assays showed high specificity for <em>M. oryzae </em>and MoT, respectively. Both assays were sensitive to ~5 pg DNA/reaction. For field application, PoT2 and MoT3 assays were tested on MoT-infected wheat seed and spikes and identified <em>M. oryzae </em>and MoT, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial <em>nad5</em> gene (NADH-dehydrogenase) was chosen as an internal control to target plant DNA. Nad5 was multiplexed with PoT2 and MoT3 and showed comparable results to individual assays. These results show applicability of these assays for MoT field surveillance, as well as identifying non-wheat species that may serve as a reservoir and/or source of inoculum for nearby wheat fields.</div>
|
