First report of leaf spot on Canada goldenrod ( Solidago canadensis ) caused by Alternaria alternata in China

Canada goldenrod (Solidago canadensis L.), a perennial Asteraceae herb native to North America, has become a globally significant invasive weed and is listed as a key management invasive species in China (Ying et al. 2025). In June 2025, dark brown spots were observed on the leaves of Canada goldenrod in Chibi (29.72ºN, 113.88ºE), Hubei Province, China. Investigations covering 0.1 hectares revealed that the disease incidence stood at 10%. Initially, small, dark-brown lesions appeared on the leaf margin. As the symptoms progressed, necrotic lesions expanded or fused into large, dark brown spots with irregular shape. Three diseased leaves were collected and transported to Yangtze University for pathogen isolation and purification. Five pieces (5 mm × 5 mm) from the junction between diseased and healthy leaf tissue were sampled, and were surface-sterilized (75% ethanol, 15 s; 1.5% NaClO, 2 min), triple-rinsed in sterile water, and were placed on potato dextrose agar (PDA) plates. The plates were stored at 25 °C in dark. After 7 days, five colonies with similar morphological characteristics were obtained on PDA and two representative isolates SCG1 and SCG2 were used for further study. The mycelia of SCG1 and SCG2 on PDA initially were white, which were turned light brown after 3 days, then were gradually turned black brown after 7 days. The fungal colonies were olivaceous to dark olive, wool-like, with whitish mycelium borders. Conidia were brown, drumstick-like or clavate, 1 - 5 transverse and 0 - 3 longitudinal septa, with darker in septa, usually 14.36 - 45.96 × 6.96 - 19.40 μm in size (n=100). The isolates were identified as Alternaria sp. (Simmons 2007) based on the morphological characteristics. For molecular identification, genomic DNA was extracted, and six loci were amplified/sequenced: TEF1-α (EF1-728F/EF1-986R), ITS (ITS1/ITS4), EndoPG (PG3/PG2b), RPB2 (RPB2-5F2/RPB2-7CR), Alt a 1 (Alt-for/Alt-rev), and GAPDH (gpd1/gpd2) (White et al. 1990; Woudenberg et al. 2015). The obtained sequences were deposited in GenBank (TEF1: PX624130, PX624131; ITS: PX610936, PX610937; EndoPG: PX624128, PX624129; RPB2: PX624126, PX624127; Alt a 1: PX624124, PX624125; GAPDH: PX624132, PX624133). BLASTn results revealed that the sequences of TEF1, ITS, EndoPG, RPB2, Alt a 1, and GAPDH of isolate SCG1 had 100%, 98.83%, 99.28%, 99.26%, 100%, and 99.82%, sequence identity to the ex-type strain A. alternata CBS 916.96 (KC584634, KF465761, JQ811978, KC584375, AY563301, AY278808), respectively. The isolate SCG2 had 99.55%, 100%, 99.32%, 99.19%, 100%, and 99.82% respective sequence identity. A phylogenetic tree was constructed using the neighbor-joining method (bootstrap= 0.005) in MEGA 7. Based on the morphological characteristics and molecular analysis, SCG1 and SCG2 were identified as A. alternata. Pathogenicity test were conducted as follows: Leaves from five healthy Canadian goldenrod plants were inoculated with SCG1 isolate conidial suspension (1.0×105 conidia/mL). Five healthy control plants were sprayed with sterile water. All plants were maintained in a greenhouse at 25 °C, 80% relative humidity, under a 12 h light/dark cycle. The experiment was repeated three times. Dark brown lesions similar to those in the samples were appeared on test leaves 7 days after inoculation, but no symptoms were observed on the control leaves. The same fungus was reisolated from the diseased leaves based on morphological characterization and sequence analyses, but not from the healthy controls, which fulfilled Koch's postulates. To our knowledge, this is the first report of A. alternata infected Canada goldenrod in China, which will provide a theoretical basis for the biological control of this invasive weed.