Fig. 1

Key parameters evaluated to establish a representative Hordeum vulgare seed lot sample for Ustilago nuda qPCR detection. The parameters included: (A) the number of seeds tested (2000 versus 7000), (B) the amount of flour used for extraction (0.02 g versus 0.2 g), and (C) the number of flour batches used for the ten DNA sub-sample extractions (i.e. one batch extracted ten times or ten batches extracted one time each). The later analysis was used to assess inter- and intra-flour batch U. nuda DNA variation. The parameters in (A) and (B) were assessed with ten DNA extractions, each from a separate flour batch. U. nuda infection levels were quantified with the qPCR method developed in this study using the ratio U. nuda to H. vulgare DNA, which normalizes the pathogen DNA to its host DNA. Three different H. vulgare seed lots, each of a different cultivar, are shown in different colors. Kruskal-Wallis tests showed no significant differences in the detected U. nuda infection levels between each of the three key parameters within cultivars.