First report of leaf blight caused by Alternaria tectorum on Pseudostellaria heterophylla in China

Pseudostellaria heterophylla (Miq.) Pax (Caryophyllaceae), known for its immunomodulatory properties, is a high-value medicinal plant widely cultivated in China (Qin et al., 2025). In June 2024, a severe outbreak of leaf blight was observed throughout the P. heterophylla cultivation base (approximately 0.5 ha field) located in Shibing County, Guizhou Province, China (27°2′4″N, 108°7′26″E). The disease incidence was 70%-75%, causing significant economic losses. The disease symptoms initially appeared as interveinal/marginal chlorotic lesions, progressed to irregular dark-margined necrotic spots, and then turned to wilting. To identify the pathogen, symptomatic leaf tissues were collected from ten diseased plants. Symptomatic tissues (5 × 5 mm) from lesion margins were surface-sterilized (75% ethanol for 30 s, 2% NaClO for 1 min, followed by three sterile-water rinses) and plated on potato dextrose agar (PDA). After incubation at 28°C in darkness for 3 days, 15 morphologically consistent fungal isolates were obtained. The fungal colonies were initially gray-white with cottony aerial hyphae. In a later stage, they became dark gray with prominent production on the reverse side. Microscopic analysis revealed that the septate conidiophores (5.3 to 35.8 × 3.2 to 15.6 μm; n=50) were straight to slightly curved, ranging from pale to dark brown in color. These morphological characteristics were consistent with descriptions of Alternaria tectorum (Gou et al., 2022). Three representative isolates (A11, A21, and A31) were selected for molecular identification by amplifying the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF), RNA polymerase II second largest subunit (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions by ITS1/ITS4 (White et al., 1990), TEF1-728F/TEF1-986R (Carbone and Kohn 1999), RPB2-5F2/fRPB2-7cR (Sung et al., 2007), and gpd1/gpd2 (Berbee et al., 1999) primers, respectively. The sequences were deposited in GenBank (ITS: PV687528, PV687529, and PV687530; TEF-1α: PV699884, PV699887, and PV699890; RPB2: PV699883, PV699886, and PV699889; GAPDH: PV699882, PV699885, and PV699888). BLAST analysis of above sequences showed 98.14 to 99.80% identities to the type-material A. tectorum ex-type YZU 161050. Then, a neighbor-joining phylogenetic tree constructed using MEGA 11 based on concatenated sequences of ITS, TEF-1α, RPB2, and GAPDH confirmed that the isolates (A11, A12, and A31) were A. tectorum. Koch’s postulates were met to confirm the pathogenicity of three isolates on P. heterophylla leaves by inoculating three-month-old plant with 1 × 10⁶ conidia/mL. Meanwhile, sterilized distilled water was employed as the control. The experiments were conducted three times with six plants in each treatment. The plants were kept in a greenhouse at 23±2℃ and 85% relative humidity. After 7 days, all leaves inoculated with A11, A21, or A31 developed necrotic lesions identical to field symptoms, whereas controls remained healthy. The pathogens were reisolated from symptomatic tissues and confirmed as A. tectorum by morphological and molecular analyses mentioned above. Until date, different Alternaria spp species have been reported as leaf blight pathogens on P. heterophylla (Wang et al., 2023; Yao et al., 2024). To our best of knowledge, this study is the first report of A. tectorum as the causal agent. The identification of the pathogen could provide background for its future management.