Detection and Quantification of <i>Serpula himantioides</i> in the Wood of <i>Chamaecyparis pisifera</i> Butt Rot Trees by Real-Time PCR

Abstract

Serpula himantioides 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 S. himantioides in the wood of Sawara cypress (Chamaecyparis pisifera) 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 S. himantioides. The specificity of the designed primer set was tested by end-point PCR and amplicon sequencing. End-point PCR assays were positive for S. himantioides and negative for S. lacrymans, which belongs to the same genus as S. himantioides, Coniophora puteana of the same family as S. himantioides, and other wood decay fungi. Taxonomy assignment based on amplicon sequencing detected only Serpula spp., and most of them were identified as S. himantioides. The linearity of the calibration curve for absolute quantification by real-time PCR assays was confirmed in the range from 101 to 108 copies. This molecular assay method using real-time PCR could detect trace amounts of S. himantioides in decayed wood, showing the applicability for early diagnostics of butt rot emergence in forests.

Keywords

• <i>Serpula himantioides</i>
• fungi
• early diagnostics
• environmental sample
• real-time PCR
• amplicon sequencing