Fig 1.
Ergot sclerotia observed in sample 9129 (Rye).
Sclerotia are indicated by arrows. This sample had an ergot severity rating of 0.294% on a percentage weight basis (Table 2). Scale bar indicates 1 cm.
Table 1.
Seed samples selected for microbiome profiling using cpn60.
Table 2.
Quantification of C. purpurea DNA in grain wash samples using ITS-targeted ddPCR and cpn60-targeted LAMP.
Table 3.
ITS and cpn60 clone diversity observed in sclerotia sourced from Manitoba, Canada.
Fig 2.
Phylogenetic analysis of cpn60 UT sequences derived from microbial profiling and ergot sclerotia compared to reference sequences.
Sequences are prefixed by cpnDB ID number (www.cpndb.ca) and GenBank accession numbers (www.ncbi.nlm.nih.gov) are provided in parentheses where available. The tree was calculated using the Maximum Likelihood method based on the Tamura-Nei model [22] using MEGA6 [23]. 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.
Fig 3.
ITS-targeted qPCR assay linearity assessed by standard curve (A) or ddPCR calibration curve (B).
Table 4.
Spearman rank correlation between ergot severity (% weight basis) and molecular quantification of ergot DNA in grain wash templates.
Fig 4.
cpn60-targeted LAMP assay linearity assessed by expressing Tp related to C. purpurea genome copies using the two LAMP detection systems evaluated in this study.
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).
Table 5.
Intra-assay reproducibility of the cpn60-targeted LAMP (isothermal detection).
Table 6.
Sensitivity and specificity of the C. purpurea cpn60-targeted LAMP assay compared to visual rating (gold standard).