Status of Dutch Elm Disease Fungus Ophiostoma novo-ulmi and Assessment of Its Temporal Variability in Croatia.

Dutch elm disease (DED) was originally caused by the ascomycete Ophiostoma ulmi, which has been replaced by a more virulent species, O. novo-ulmi, divided into subsp. novo-ulmi and subsp. americana. Permeable reproductive barriers, a period of co-occurrence of O. ulmi and O. novo-ulmi, and the current overlap of O. novo-ulmi subspecies have been important in shaping the present O. novo-ulmi populations in Europe, which were initially clonal, predominantly of the MAT-2 type. This study confirmed the persistence of O. novo-ulmi in Croatia over the years, although at some forest sites, the diseased elms were not detected. The methodology used to assess changes in O. novo-ulmi populations was based on the col1 and cu genes, which have subspecies-specific nucleotide differences, analysis of MAT idiomorphs, and temperature-growth responses. The col1 and cu gene sequencing did not reveal a change in the number of isolates with the recombinant col1/cu genotype over 10 years (2012-2022). At both sampling times, approximately one-fourth of all analyzed isolates had recombinant col1/cu genotypes. However, the frequency of MAT-1 isolates, which all have MAT-1 genes originating from O. ulmi, increased during this period. Differences in growth rate at 20, 26, and 30 °C revealed variations in the temperature response of isolates, which were affected by sampling time and mating type. The MAT-1 isolates were shown to grow more slowly than MAT-2 at the three temperatures tested. The advantage of MAT-2 was reflected in temporal differences in growth rate at resampled sites, particularly at lower temperatures. These results suggest that changes in the frequency of mating types in Croatia occurred between 2012 and 2022, accompanied by modifications in the pathogen's response to temperature at the population level.