First Report of Leaf Spot Disease Caused by Alternaria alternata on Pepper in Hunan Province, China

Pepper (Capsicum annuum L.) is an important cash crop widely cultivated in China (Zou et al. 2026). In April 2026, leaf spot was detected in a pepper field at the Hunan Academy of Agricultural Sciences (28°12′27″N, 113°05′34″E), Changsha, Hunan Province. Initial small, dark spots enlarged into irregular pale brown necrotic lesions with dark brown margins and concentric rings; severely affected plants yellowed and were stunted. Eight symptomatic leaves were collected from two plants. After rinsing with sterile distilled water and blotting dry, 3 × 3-mm pieces from lesion margins were surface-disinfested in 75% ethanol for 30 s and 10% sodium hypochlorite for 60 s, rinsed three times with sterile water, plated on potato dextrose agar (PDA) amended with 150 μg/ml chloramphenicol and kanamycin, and incubated at 28°C for 3 days (Hu et al. 2026). Mycelia were transferred to fresh PDA and incubated at 28°C for 3 to 5 days. Colonies were initially grayish green with white floccose mycelia and a white margin, becoming dark brown with dense, cottony aerial mycelia. Conidia were pale to dark brown, obpyriform or ellipsoidal, with 0 to 5 transverse and 0 to 2 longitudinal septa; some bore a short cylindrical apical beak. Conidia measured 9.5 to 33.3 × 3.7 to 13.1 µm (n = 100), consistent with Alternaria spp. For molecular identification, the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (gpd), RNA polymerase II second-largest subunit (rpb2), and translation elongation factor 1-alpha (tef1) genes were amplified with ITS4/ITS5 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F2/fRPB2-7cR (Sung et al. 2007), and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. A BLAST search showed that these sequences had 100% identity with corresponding sequences of Alternaria alternata (ITS, AF347032; gpd, AY278809; rpb2, KC584435; tef1, KC584693). The sequences were deposited in GenBank (ITS: PZ506518; gpd: PZ529550; rpb2: PZ529551; tef1: PZ529549). A concatenated phylogenetic tree based on the four genes showed that the isolate clustered significantly with Alternaria alternata. For pathogenicity tests, leaves of three pepper plants were sprayed to runoff with a conidial suspension (1 × 10⁶ conidia/ml); three control plants received sterile distilled water with 0.1% Tween 80. Plants were covered with transparent plastic bags for 24 h and then maintained at 28°C, 80% relative humidity, and a 12-h photoperiod. After 10 days, inoculated plants developed symptoms similar to those observed in the field, whereas controls remained symptomless. The fungus was reisolated from symptomatic leaves and identified as A. alternata by morphology and sequencing, fulfilling Koch’s postulates. Alternaria alternata has been reported as a major causal agent of leaf spot on other plants, such as Tetrastigma hemsleyanum (Ren et al. 2025) and Buxus harlandii (Zhang et al. 2025). To our knowledge, this is the first report of A. alternata causing leaf spot disease on pepper in Hunan Province, China. This finding provides a basis for disease diagnosis and management.