First Report of Alternaria alternata Causing Leaf Spot on Curcuma involucrata in China

Curcuma involucrata (King ex Baker) Škorničk. (syn. Stahlianthus involucratus (King ex Baker) Craib), a perennial Zingiberaceae herb known as “Tu Tian Qi” in Chinese, has been traditionally used to relieve muscle pain and edema (Pingsusaen et al., 2015). In October 2025, a severe leaf spot outbreak was observed on C. involucrata in an herbal nursery in Kunming, Yunnan Province, China (24.84°N, 102.83°E). Initial symptoms appeared as small yellow spots with chlorotic halos, which progressively enlarged into elongated dark brown lesions, eventually causing extensive blighting. Symptomatic leaf tissues (5 × 5 mm) from three leaves of five plants were surface-sterilized with 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min, washed three times with sterile water, then plated on potato dextrose agar and incubated at 28°C in the dark for 3 days. A predominant fungus was isolated from 85% of the samples. Single-spore purification of this fungus yielded six morphologically uniform isolates. Colonies were densely villous, grayish-black to dark brown on the surface, with the reverse showing a dark brown center fading to pale yellowish-brown toward the margins. Conidia were pale to dark brown, obclavate or ovoid, with both transverse and longitudinal septa, measuring 22.5 to 40 μm × 8 to 13.5 μm (n = 30), borne on short hyaline to pale brown conidiophores. Genomic DNA was extracted from mycelia of two representative isolates using the CTAB method. The internal transcribed spacer (ITS), 28S large subunit rRNA (LSU), 18S small subunit rRNA (SSU), and RNA polymerase II second largest subunit (RPB2) were amplified with primer pairs ITS1/ITS4 (White et al., 1990), LR0R/LR5 (Schoch et al., 2012), NS1/NS4 (White et al., 1990), and fRPB2-5F/fRPB2-7Cr (Liu et al., 1999), respectively, then sequenced and assembled. Both isolates shared identical sequences, and those of SiF01 were deposited in GenBank. BLASTn showed that the ITS (GenBank accession no. PZ586753), LSU (PZ586763), SSU (PZ586757), and RPB2 (PZ600164) sequences of SiF01 were 99.4, 99.8, 99.9, and 100% identical to Alternaria alternata strain CBS 916.96 (AF347031, DQ678082, KC584507, and KC584375), respectively. A maximum likelihood phylogenetic tree was constructed based on the ITS, LSU, SSU, and RPB2 concatenated sequences using MEGA X. SiF01 clustered with A. alternata. Based on morphology and multilocus sequence analyses, SiF01 was identified as A. alternata (Woudenberg et al., 2013). To confirm pathogenicity, 1-year-old C. involucrata plants (approximately 20 cm tall) were sprayed to runoff with a conidial suspension of SiF01 (1 × 10⁶ conidia/ml). Controls were sprayed with sterile water. Plants were kept at 25°C with a 12-h photoperiod and 80% relative humidity. After 14 days, inoculated leaves developed yellowish-brown to dark-brown necrotic lesions with yellow halos, consistent with the field symptoms. A. alternata, confirmed by morphology and ITS sequencing, was reisolated from inoculated leaves but not from control leaves, fulfilling Koch’s postulates. The experiment was repeated three times with three plants per treatment. A. alternata has been reported to cause leaf spot on other medicinal plants cultivated in Yunnan Province, including Paris polyphylla var. yunnanensis, Polygonatum kingianum, and Panax notoginseng. To our knowledge, this is the first report of A. alternata leaf spot on C. involucrata in China, providing a basis for developing targeted disease management strategies and preventing potential yield losses in this cultivated medicinal herb.