Rapid and accurate prediction of the frequencies of Bursaphelenchus xylophilus and B. mucronatus in mixed nematode samples using real-time species-specific PCR

Accurate detection of Bursaphelenchus xylophilus and prediction of its frequency in crude nematode samples is often hindered by the coexistence of related nematode species, such as B. mucronatus, that are morphologically similar but non-pathogenic. To establish a detection system enabling determination of the relative frequencies of B. xylophilus and B. mucronatus from field nematode samples, we developed a real-time species-specific PCR (rtssPCR) protocol which targets the substantial sequence differences in the 5S rRNA marker gene between the two nematode species. Using standard DNA mixtures of B. xylophilus and B. mucronatus in various ratios, plots of percent species proportion vs cycle threshold value (Ct value) were generated for the prediction of species frequency. The rtssPCR protocol enables the detection of target nematode frequencies as low as 0.16% at the 95% confidence level. When nematode DNA samples were extracted from the mixed specimens of B. xylophilus and B. mucronatus in various ratios and analysed by rtssPCR, the semi-log plot was nearly identical to the plot generated from standard mixed DNA samples, demonstrating that field populations of the nematodes can be directly used for rtssPCR analysis. The rapid and accurate determination of B. xylophilus or B. mucronatus frequencies by this rtssPCR protocol makes it ideal for routine monitoring and quarantine of B. xylophilus in the field.