| Summary: | <i>Serpula himantioides</i> is a wood-decaying fungal pathogen which is widespread and causes potentially serious butt rot in living trees of numerous coniferous plantation species. This study aimed to quantify <i>S. himantioides</i> in the wood of Sawara cypress (<i>Chamaecyparis pisifera</i>) with butt rot using real-time polymerase chain reaction (real-time PCR). Species–specific primers were designed for the internal transcribed spacer 2 (ITS2) regions of ribosomal DNA (rDNA) of <i>S. himantioides</i>. The specificity of the designed primer set was tested by end-point PCR and amplicon sequencing. End-point PCR assays were positive for <i>S. himantioides</i> and negative for <i>S. lacrymans,</i> which belongs to the same genus as <i>S. himantioides</i>, <i>Coniophora puteana</i> of the same family as <i>S. himantioides</i>, and other wood decay fungi. Taxonomy assignment based on amplicon sequencing detected only <i>Serpula</i> spp., and most of them were identified as <i>S. himantioides</i>. The linearity of the calibration curve for absolute quantification by real-time PCR assays was confirmed in the range from 10<sup>1</sup> to 10<sup>8</sup> copies. This molecular assay method using real-time PCR could detect trace amounts of <i>S. himantioides</i> in decayed wood, showing the applicability for early diagnostics of butt rot emergence in forests.
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