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

Sensitivity and specificity of the <i>C</i>. <i>purpurea cpn60</i>-targeted LAMP assay compared to visual rating (gold standard).

<p>Sensitivity and specificity of the <i>C</i>. <i>purpurea cpn60</i>-targeted LAMP assay compared to visual rating (gold standard).</p

Spearman rank correlation between ergot severity (% weight basis) and molecular quantification of ergot DNA in grain wash templates.

<p>Spearman rank correlation between ergot severity (% weight basis) and molecular quantification of ergot DNA in grain wash templates.</p

Ergot sclerotia observed in sample 9129 (Rye).

<p>Sclerotia are indicated by arrows. This sample had an ergot severity rating of 0.294% on a percentage weight basis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173495#pone.0173495.t002" target="_blank">Table 2</a>). Scale bar indicates 1 cm.</p

<i>cpn60</i>-targeted LAMP assay linearity assessed by expressing T_p related to <i>C</i>. <i>purpurea</i> genome copies using the two LAMP detection systems evaluated in this study.

<p>The equations for each curve are: y = -9.03x+96.98 (calcein detection) and y = -1.90x+19.44 (isothermal detection). The correlation coefficients (r²) are 0.99 (calcein detection) and 0.95 (isothermal detection).</p

ITS-targeted qPCR assay linearity assessed by standard curve (A) or ddPCR calibration curve (B).

<p>ITS-targeted qPCR assay linearity assessed by standard curve (A) or ddPCR calibration curve (B).</p

Phylogenetic analysis of <i>cpn60</i> UT sequences derived from microbial profiling and ergot sclerotia compared to reference sequences.

<p>Sequences are prefixed by cpnDB ID number (<a href="http://www.cpndb.ca" target="_blank">www.cpndb.ca</a>) and GenBank accession numbers (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>) are provided in parentheses where available. The tree was calculated using the Maximum Likelihood method based on the Tamura-Nei model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173495#pone.0173495.ref022" target="_blank">22</a>] using MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173495#pone.0173495.ref023" target="_blank">23</a>]. The tree was bootstrapped (100 iterations) and numbers next to the nodes indicate the percentage of trees in which the associated taxa clustered together. Branch lengths correspond to the number of substitutions per site.</p

Seed samples selected for microbiome profiling using <i>cpn60</i>.

<p>Seed samples selected for microbiome profiling using <i>cpn60</i>.</p

Quantification of <i>C</i>. <i>purpurea</i> DNA in grain wash samples using ITS-targeted ddPCR and <i>cpn60</i>-targeted LAMP.

<p>Quantification of <i>C</i>. <i>purpurea</i> DNA in grain wash samples using ITS-targeted ddPCR and <i>cpn60</i>-targeted LAMP.</p

ITS and <i>cpn60</i> clone diversity observed in sclerotia sourced from Manitoba, Canada.

<p>ITS and <i>cpn60</i> clone diversity observed in sclerotia sourced from Manitoba, Canada.</p