Quantitative molecular diagnostic assays of grain washes for <i>Claviceps purpurea</i> are correlated with visual determinations of ergot contamination
<div><p>We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (<i>cpn60</i>). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined <i>cpn60</i> sequences from field-collected and reference strains of the ergot fungus, <i>Claviceps purpurea</i>. These data allowed us to identify this fungal sequence as deriving from <i>C</i>. <i>purpurea</i>, and suggested that <i>C</i>. <i>purpurea</i> DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of <i>C</i>. <i>purpurea</i> DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of <i>Claviceps</i> DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a <i>cpn60</i>-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.</p></div
