Species-Specific Primers for Eutypella parasitica, the Causal Agent of Eutypella Canker of Maple.

Abstract

Eutypella parasitica was recently reported in Europe for the first time, and this study reports the molecular evaluation of the internal transcribed spacer (ITS)1/5.8S/ITS2 regions of 68 isolates of the fungus obtained in pure culture with polymerase chain reaction restriction fragment length polymorphism (RFLP). The RFLP patterns of all isolates proved identical and the restriction profiles served to differentiate E. parasitica from Eutypa lata, another pathogenic member of the family Diatrypaceae. Low intraspecific variability was detected in the sequenced ITS1/5.8S/ITS2 regions of eight Eutypella parasitica isolates originating from different hosts and geographical locations. Based on this ITS region, EpR/F primers specific to E. parasitica were constructed and tested with a wide range of fungal isolates. The EpR/F primer pair successfully amplified the expected fragment size of 341 bp from isolates of E. parasitica and also directly from infected maple wood shavings. The RFLP patterns and species-specific primers represent a step toward routine, large-scale, and rapid molecular diagnostics and identification of E. parasitica.