First Report of Brown Spot on <i>Paris polyphylla</i> var. <i>yunnanensis</i> Caused by <i>Alternaria brassicae</i> in China

Yunnan Chonglou (Paris polyphylla var. yunnanensis) is mainly distributed in the provinces of Yunnan, Sichuan, and Guizhou, and is a shade plant. Yunnan Chonglou is an important raw material for many Chinese patent medicines, such as Yunnan Baiyao and Gong Xuening (Sun et al. 2020). It is extensively cultivated in China. In August 2022 and June 2024, brown spots were observed on leaf of P. polyphylla var. yunnanensis in Wenshan prefecture of Yunnan province, China. The planting base of P. polyphylla var. yunnanensis had different degrees of disease at Wenshan prefecture of Yunnan province (22°46 N, 103°30 E). We focused our survey on three artificial planting bases, each with an area of 1.8 hectares and 2500 P. polyphylla var. yunnanensis plants per hectare, and the incidence of the disease was found to be around 45-63%. Lesions appeared as round brown spots with green halos around them, and the skin lesions later became irregular and necrotic on the leaves. The pathogen was isolated using the single-spore separation method (Chen et al. 2022). Under a microscope, single conidia were picked using needles and transferred onto potato dextrose agar (PDA) medium. A total of seven isolates with similar morphology were obtained. The colonies were initially white, later turned brown or black, and covered the PDA plate in 6 d. The hyphae were dense and cottony. Conidia were produced in short chains of 2 to 5, and generally simple conidiophores were produced on the PDA surface. Conidia were brown to dark brown, ovoid, nearly cylindrical, with 1 to 6 transverse and 0 to 2 longitudinal septa, and 9.15 to 28.9 × 4.85 to 12.92 µm (mean 18.423 × 8.885 µm) (n = 50). The morphological features were consistent with those of Alternaria brassicae. For molecular identification, the ribosomal internal transcribed spacer (ITS) and the major Alt a 1 allergen (alt a1) gene were amplified and sequenced, respectively (Zhang et al. 2020). The sequences were deposited in GenBank (Accession: ITS: PX239442, alt a1: PX239923). Multilocus phylogenetic tree were developed by Neighbor-joining (NJ) methods, which showed that WSCL34 clustered together with the A. brassicae with 100% bootstrap support. Based on morphological characteristics and molecular analysis, the isolates were identified as A. brassicae. Thirty attached P. polyphylla var. yunnanensis leaves were inoculated with conidial suspension (1×106 spores/mL), and another 30 used as controls were inoculated with sterile water at 25°C with a 12 h photoperiod and 90% humidity. After 7 d, the leaves began to show symptoms identical to those of the disease in the field, whereas the control was asymptomatic. The pathogen A. brassicae was reisolated and identified by morphological characterization and molecular analysis after reisolation from the inoculated leaves, thus fulfilling Koch’s postulates. This is the first report of P. polyphylla var. yunnanensis brown spot disease caused by A. brassicae in China. This study is critical for advancing effective management and control practices for brown spot disease, and this finding provides a theoretical basis for the control of P. polyphylla var. yunnanensis diseases.