TaqMan PCR assay for detection and quantification of Stagonosporopsis tanaceti in pyrethrum seed and seedlings

Abstract

Pyrethrum seed has an important role in the transmission of Stagonosporopsis tanaceti, the cause of ray blight disease of pyrethrum. A TaqMan probe based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inocula in pyrethrum seed and seedlings. Primer pair (St_qF3, St_qR2) was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using St_qF3, St_qR2 and a probe St_qP was highly specific against the genomic DNA of S. tanaceti, but did not amplify DNA of 14 related Stagonosporopsis species or other foliar pathogens of pyrethrum. The sensitivity limit of this assay was measured using the cycle threshold (Ct) value which ranged from 17.59 for 10 nanograms (ng) to 36.34 for 100 femtograms (fg) genomic DNA of S. tanaceti. There was a significant negative correlation (r = −0.999, P < 0.001) between the Ct value and the percent of S. tanaceti infected seed. In addition, this TaqMan PCR assay detected latent infection within seedlings. This assay could be applied to test commercial seed and seedlings before deciding on the appropriate management practices.