Specific detection of a basidiomycete, Phlebia brevispora associated with butt rot of Chamaecyparis obtusa, by PCR-based analysis

Abstract

In order to monitor the basidiomycetous fungus Phlebia brevispora isolated from butt rot of Chamaecyparis obtusa (Japanese cypress) in 1997 in Nagasaki Prefecture, a sensitive polymerase chain reaction (PCR)-based assay was developed to specifically detect the fungus on-site. A species-specific primer for P. brevispora was derived from the internal transcribed spacer (ITS) region (containing 5.8S ribosomal DNA, ITS1 and ITS2) sequences of the fungus. The PCR assay was able to detect down to 1 fg DNA (per 1 µl PCR reaction mixture) and down to 0.2 mg mycelium of P. brevispora (per 1 g of decayed wood). The samples for on-site monitoring were collected in 2002 from the decayed tree stump in which P. brevispora had first been isolated. From the decayed tree tissue, P. brevispora could be detected by PCR assay even when its mycelium could not isolated from the tree tissue by culturing. This indicates that the PCR amplification using the specific primer developed here is a useful method for monitoring P. brevispora on-site.