Optimization of sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees

Aims: To develop fast and reliable sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees. Methods and Results: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4 mm-diameter, 43 cm long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analyzing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1-4 drillings were either analyzed separately, and the results combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56.6% for the scheme based on a single drill to 96.8% for the scheme based on four drillings analyzed separately. Both schemes significantly differ (P< 0.05) from those based on two and three drillings, whose efficiency was 72.6% and 83.9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. Conclusions: Highest diagnostic efficiency was obtained by analyzing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. Significance and Impact of Study: Fast and reliable sampling procedures make DNA-based diagnosis more suitable for tree inspection procedures.