Identification and characterization of the causal agent of Dutch elm disease in Croatia

Dutch elm disease (DED) is a vascular wilt disease of elms caused by ascomycetous fungi of the genus Ophiostoma. The initial DED pandemic was caused by Ophiostoma ulmi, a relatively weak pathogen when compared to Ophiostoma novo-ulmi, the causal agent of the current pandemic. The latter has been spreading as two distinct subspecies: O. novo-ulmi ssp. novo-ulmi and O. novo-ulmi ssp. americana. Weak prezygotic barriers to hybridization have resulted in the emergence of ssp. novo-ulmi × ssp. americana hybrids in subspecies overlap zones. In this study, samples of host species Ulmus laevis, Ulmus minor and Ulmus glabra from across Croatia were analysed for DED prevalence and pathogen distribution and characterization. Dutch elm disease was confirmed in all three elm species, but the prevalence of the infection was approximately four times higher in U. minor and U. glabra than in U. laevis. Ophiostoma novo-ulmi was identified as the only cause of DED, while O. ulmi was not detected, indicating complete replacement of O. ulmi by O. novo-ulmi in Croatia. PCR–RFLP analysis and sequencing of the cu and col1 genes indicate the presence of both O. novo-ulmi subspecies as well as their hybrids at investigated sites, often in high frequency. The results provide insight into distribution and population structure of Dutch elm disease pathogen in Croatia, as well as an assessment of virulence for selected isolates, which could be utilized for making informed management decisions regarding DED and elm conservation in Croatian forests.