First Report of the ability of Alternaria alternata to cause canker in sea buckthorn ( Hippophae rhamnoides ) in China

Sea buckthorn (Hippophae rhamnoides) is an economically important medicinal crop in Xinjiang, China (Zhu et al., 2025). In August 2024, a canker disease was observed on trunks and branches of sea buckthorn in experimental fields at Tarim University (N40°54′44.28″, E81°30′12.48″), Aral City, Xinjiang, China. Across a 0.2-ha area, 136 trees were surveyed, of which 74 trees (54.41%) showed disease symptoms. Affected trunks and branches exhibited ulceration and cracking, with dark brown necrotic lesions internally. Twelve symptomatic trunks and branches were collected from multiple trees. After surface sterilization, tissue sections were placed on PDA and incubated in darkness at 26 °C for 5 days. Five isolates, each originating from a different symptomatic tree, were obtained and showed identical cultural and morphological characteristics. Based on this morphological uniformity, One representative isolate (designated SHAJI-1) was selected for detailed characterization. After 9 days, the colonies on PDA were 77 to 84 mm in diameter. Colonies of the SHAJI-1 strain were round, greyish-green with abundant cottony aerial hyphae. The reverse side showed a circular dark brown ring with white margins. Conidiophores were brown, simple or branched, producing numerous conidia in short chains. Conidia were obclavate to ellipsoid, smooth or slightly spiny, brown, with 1-5 transverse septa and 0-3 longitudinal septa, measured 11.1-24.8 × 8.5-20.6 µm (n = 50), and were rostrate. Based on morphological features and sporulation pattern, the pathogen was identified as Alternaria alternata. For molecular identification, the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R (Carbone and Kohn, 1999), gpd1/gpd2 (Berbee et al., 1999), and RPB2-5F2/fRPB2-7cR (Liu et al., 1999), respectively. The resulting sequences were deposited in GenBank under accession numbers PV706213 (ITS), PV682933 (TEF), PV690107 (GAPDH), and PV690106 (RPB2). BLAST analysis showed 99-100% identity with the corresponding sequences of the Alternaria alternata type strain CBS 479.90: ITS (MH862229.1), TEF (KP125095.1), RPB2 (KP124787.1), and GAPDH (KP124174.1). Sequence data for various Alternaria species were obtained from the National Center for Biotechnology Information (NCBI) database, and the phylogenetic relationship of the SHAJI-1 strain was analyzed for comparative purposes. Based on morphology and phylogeny (ITS, TEF, GAPDH, RPB2; ML; Fig. S2), the fungus was identified as Alternaria alternata. Pathogenicity tests were conducted on 1-year-old healthy branches of sea buckthorn. A wound (ca. 0.5 mm) was made using a sterilized punch. Six branches received a PDA plug of SHAJI-1; Six control branches received sterile plugs. Inoculation sites were covered with wet cotton and Parafilm. Experiments were conducted in a greenhouse (26 °C, 60 % RH, 12-h photoperiod). After 10 days, cankers developed (mean vascular discoloration length 79.38 mm). The experiment was repeated three times. Koch’s postulates were fulfilled after re-isolation and confirmation of the fungus; Controls remained asymptomatic. First report of A. alternata causing canker on sea buckthorn in China and globally, providing critical information for disease etiology and management.