Long-term preserved mycelium establishes the presence of Cryphonectria carpinicola in the Balkans and of Cryphonectria radicalis in Bulgaria Carolina Cornejo & Mihajlo Risteski & Kiril Sotirovski & Daniel Rigling Accepted: 18 October 2022 # The Author(s) 2022 Abstract Around the world, isolate collections in my- cological institutes have long preserved valuable speci- mens and information for science. In the present study, we determine Cryphonectria-like taxonomic rarities in our 10–24 years old isolate collection from the Balkans to link preserved dry mycelium with DNA data. Using ITS sequences, we confirm for the first time the occur- rence of Cryphonectria carpinicola on a Carpinus tree in the Balkans and extend the range of Cryphonectria radicalis found on Castanea sativa to Bulgaria. The oldest specimen examined dates from 1998 and molecu- larly confirms the first reported finding of Cryphonectria radicalis for North Macedonia. Keywords Fagaceae . Betulaceae . Tree pathogen . Species diversity . Barcoding . Culture collection Members of the ascomycetous genus Cryphonectria (Sacc.) Sacc. & D. Sacc. are known to infect important deciduous tree species in the Fagaceae and Betulaceae. Cryphonectria parasitica (Murrill) M.E. Barr causes chestnut blight disease and is the best-studied Cryphonectria species. This fungus, native to East Asia, invaded North America and Europe in the last century, where it caused pronounced symptoms in Castanea Mill. species and severely damaged chestnut stands in eastern U.S.A. and in all major chestnut areas of Europe (Rigling & Prospero, 2018). For this reason, most re- search has focused on C. parasitica, while other Cryphonectria species have been overlooked and there- fore poorly studied. Examples of neglected species in Europe are Cryphonectria naterciae M.H. Bragança, E. Diogo & A.J.L. Phillips (Bragança et al., 2011) and Cryphonectria carpinicola D. Rigling, T. Cech, Cornejo & L. Beenken (Cornejo et al., 2021), which have only recently been recognized as species at all. However, little is known about their distribution range in Europe. To date, C. naterciae has been reported in Portugal, Italy, and Algeria only, and Cryphonectria carpinicola in a few Central European countries (Austria, Italy, Germany, Switzerland) and in the Republic of Georgia on the bark of dead Carpinus L. species (for more details, Cornejo et al., 2021). While investigating the invasion dynamics of C. parasitica in the Balkans in the early 2000s, we have conducted several extensive sampling campaigns of symptomatic Castanea sativa Mill. in the Balkan Moun- tains in Bulgaria, North Macedonia, and Kosovo (Fig. 1A). During isolation of fungal strains from bark material, we encountered morphologically atypical mycelia, distinct from C. parasitica, suggesting that dif- ferent Cryphonectria species may be present in this re- gion. These taxonomic rarities were preserved as dried mycelium at the plant pathology lab of the Hans Em Eur J Plant Pathol https://doi.org/10.1007/s10658-022-02613-8 C. Cornejo (*) : D. Rigling Swiss Federal Research Institute WSL, Zuercherstrasse 111, 8903 Birmensdorf, Switzerland e-mail: carolina.cornejo@wsl.ch M. Risteski : K. Sotirovski Ss. Cyril and Methodius University in Skopje, Hans Em Faculty of Forest Sciences, Landscape Architecture and Environmental Engineering, ul. 16 Makedonska brigada br. 1, MK-1000, Skopje, Republic of North Macedonia