Development of a Novel Multiplex TaqMan Probe qPCR Assay Distinguishing Dikaryotic ITS rDNA Types of <i>Sclerotium rolfsii</i> (<i>=Agroathelia rolfsii</i>) and <i>S. delphinii</i>

<i>Sclerotium</i> <i>rolfsii</i> (=<i>Agroathelia rolfsii</i>) and <i>S. delphinii</i> are globally ubiquitous and prevalent soilborne pathogens. These species are distinguishable by the morphology of their sclerotia formed on artificial media. However, this distinction requires prior pure isolation, complicating direct identification from diseased plants or soil samples. Additionally, each species contains two distinct internal transcribed spacer (ITS) ribosomal DNA types in a dikaryotic cell, further complicating their identification through sequence analysis. To address this issue, we developed a novel multiplex qPCR method utilizing TaqMan probes and primers. This method specifically targets the two ITS types unique to each species, effectively distinguishing between these pathogens and differentiating them from nontarget soilborne fungal and oomycete pathogens. Its efficiency and sensitivity were confirmed through their detections in artificially inoculated plants and soil samples. Our multiplex qPCR technique offers a precise and reliable approach for the simultaneous detection and differentiation of <i>S. rolfsii</i> and <i>S. delphinii</i>, significantly improving disease surveillance and facilitating timely, effective disease management.