| Summary: | <i>Sequoiadendron giganteum</i> Lindl. [Buchholz] is a long-lived tree species endemic to the Sierra Nevada Mountains in California. Due to its massive size and beauty, <i>S. giganteum</i> is a popular ornamental tree planted in many parts of the world, including Europe. Since 2017, scattered branch die-back has been observed on <i>S. giganteum</i> trees in Zagreb, Croatia. Other symptoms included resinous branch cankers, reddish-brown discoloration of the sapwood and, in severe cases, crown die-back. Branches showing symptoms of die-back and cankers were collected from six <i>S. giganteum</i> trees in Zagreb and the aim of this study was to identify the causal agent of the disease. The constantly isolated fungi were identified using morphology and phylogenetic analyses based on the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA), and partial sequencing of two housekeeping genes, i.e., translation elongation factor 1-α (TEF 1-α), and β tubulin 2 (TUB2). The fungi were identified as <i>Botryosphaeria dothidea</i> (Moug.) Ces. and De Not. and <i>Neofusicoccum yunnanense</i> G.Q. Li & S.F. Chen. The pathogenicity test was conducted in a plant growth chamber on <i>S. giganteum</i> seedlings and revealed that <i>N. yunnanense</i> was more aggressive compared to <i>B. dothidea. N. yunnanense</i> was able to reproduce symptoms of canker and die-back and kill plants seven weeks after inoculation whereas <i>B. dothidea</i> produced cankers. To the best of our knowledge, this is the first report of <i>B. dothidea</i> and <i>N. yunnanense</i> causing canker and die-back disease of <i>S. giganteum</i> in Croatia. It is also the first record on the identity and pathogenicity of any fungal species associated with <i>S. giganteum</i> in this country. The study expended the known host range of <i>N. yunnanense</i> to include <i>S. giganteum</i>, which is a valuable ornamental tree in Croatian landscapes. Disease management strategies should be developed to mitigate or reduce the impact of the disease.
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