Isolation and identification of monokaryotic strains of P. fraxinea.

To isolate monokaryotic strains of P. fraxinea, basidiospores of P. fraxinea were collected from a fruiting body grown on a Robinia pseudoacacia tree on the campus of Hokkaido University, Sapporo, Japan, and spread onto potato dextrose agar (PDA; Nihon Pharmaceutical Co., Ltd., Tokyo, Japan). The colonies were separated and grown several times until five single filamentous fungi were isolated (P. fraxinea strains SS1 to SS5). The internal transcribed spacer (ITS) region, including 5.8 rRNA, was amplified by a colony PCR method using Kod Fx polymerase (Toyobo Co., Ltd., Osaka, Japan). Primer set ITS1 and ITS4 was used as previously described (39). The ITS sequences were searched using NCBI BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), and all of the best hits showed >99% identity to Vanderbylia fraxinea (synonym of P. fraxinea) (see Table S1 in the supplemental material). Phylogenetic tree analysis (1,000 bootstraps) was performed with the BLASTn best-hit ITS sequences of five identified single spores (Table S1) and the previously reported ITS sequences of P. fraxinea (11, 15) by using MEGA7 software (40).