First report of canker caused by a Sphaerulina sp. on European hornbeam ( Carpinus betulus ) in the United States

Between 2022 and 2025, branch samples from fastigiate European hornbeams showing severe canopy dieback were submitted for diagnosis from multiple U.S. states (CT, MA, NY, NJ, OH, and PA). Field symptoms included sunken cankers on twigs, branches, and trunks as well as chlorotic, diffuse leaf spots. The symptomatic branch samples were examined at the Bartlett Tree Experts Diagnostic Laboratory in Charlotte, NC. Conidia from conidiomata on branches were on average 42.9 (21.9-58.5) x 3.6 (2.7-4.6) μm with 0-5 septa (n =30). Isolations from branch and twig cankers were made by placing 2-3 mm long slivers of affected tissue onto water agar. After 7 days, pure cultures were established by transferring hyphal tips onto ¼-strength acidified PDA (APDA). Culture colonies, conidiomata formed on branch tissue, and symptoms were consistent with what is reported of other Sphaerulina caused diseases on woody hosts (Ujat and Nakashima 2024). DNA was extracted from mycelium of pure cultures, and ITS and TEF1-α were amplified by PCR using primers ITS1F/ITS4 (White et al. 1990) and EF1728/EF2 (Ujat and Nakashima 2024). Purified PCR products were sequenced and resulted in 97 and 86% identity match with Sphaerulina pelargonii (NR_137936 and KP004506), respectively. Based on a phylogenetic analysis of the concatenated ITS and TEF1-α sequences, the Sphaerulina species causing canker on hornbeam appears to be a distinct species that is not presently known from DNA sequence data. All ITS and TEF1-α sequences have been deposited into NCBI GenBank (PZ221902-PZ221914: ITS; PZ238680-PZ238692: TEF1α), and representative cultures in CBS, Utrecht, Netherlands (CBS 155003-155006). Twelve (exp. 1) and nine (exp. 2) approximately two-year-old, disease-free, 2-3-foot Carpinus betulus ‘Fastigiata’ in 2-gallon pots were inoculated in the greenhouse on May 20, 2025 (exp. 1) and June 11, 2025 (exp. 2). Stems were disinfected with 70% ETOH before making a shallow bark slit (7-10 mm) into vascular cambium with a sterile scalpel approximately 25 cm above the graft on each tree. A three-week-old colonized ¼-strength APDA plug (3 mm) of isolate IG1-2 (PZ221906) on a strip of moistened Kimwipe was positioned onto the exposed cambium. The Kimwipe was wrapped around the stem and then wrapped with Parafilm. Control trees were mock inoculated similarly using non infested ¼-strength APDA plugs. On October 2, 2025 (exp. 1) and December 5, 2025 (exp. 2), trees were evaluated and all inoculated trees showed sunken, reddish discolored bark, and swollen margins around the inoculation site. Lesion length of inoculated trees averaged 26.6 mm, while mock inoculated trees showed no canker symptoms or lesions. Sphaerulina sp. was successfully reisolated from 9 out of 13 fungal inoculated trees and not recovered from control trees. Although we observed the same Sphaerulina sp. causing leafspots on hornbeam, Koch’s postulates for leaf pathogenicity remain to be completed. This is the first report of Sphaerulina canker on European hornbeam. In the last thirty years, fastigiate hornbeams have become very popular in the landscape trade as hedge trees. This disease is likely more widespread than reported here and it may affect the future suitability of this hedge tree in landscapes of the eastern United States. Future studies need to investigate the etiology of this disease as well as resolving the taxonomy of this unique Sphaerulina sp.