First Report of Leaf Spotting Disease Caused by Plectosphaerella alismatis of Alisma (Alisma plantago-aquatica Linn.) in China.

Alisma (Alisma Plantago-Aquatica Linn.) is a traditional medicinal aquatic plant in China. Since August 2024, a leaf spotting disease of Alisma, with an average incidence of 70% ~ 90% across fields, has been observed in Yucheng District, Ya`an City, Sichuan Province, China (29°98'N and 102°98'E). The round red-brown spots were first observed on the outer leaves, and then the larger spots merged, resulting in leaf yellowing and death, ultimately leading to approximately 35% yield loss. To isolate pathogenic microorganisms, ten symptomatic leaf samples were collected and disinfected with 75% ethanol for 30 s, followed by 0.5% NaClO for 2 min. After rinsing three times with sterile water, the samples were transferred to potato dextrose agar (PDA) medium supplemented with 50 mg L^-1 streptomycin sulfate, and cultured in the dark at 28°C. After 7 days, the hyphal tips of the emerging colonies were transferred to fresh PDA for purification. The eight isolates obtained were named ZX1 to ZX8. We initially tested the isolates' pathogenicity on detached leaves. 5 mm PDA agar blocks of the isolates were inoculated onto healthy detached leaves, with blank PDA agar as the control. After 7 days, only the leaves inoculated with ZX6 showed spots and yellowing symptoms similar to those observed in the field. The leaves inoculated with other isolates and the control showed no symptoms. Consequently, we further tested the pathogenicity of ZX6 on whole plants. Healthy Alisma seedlings (2-month-old) were inoculated with ZX6 conidial suspension (10⁶ spores/ml) and with clean water as the control. Plants were incubated in a growth chamber (22-28°C, 85% relative humidity, 16 h light and 8 h dark). The pathogenicity test was repeated twice. Typical leaf spot symptoms were observed on the inoculated leaves after 7 days. The pathogens, reisolated from infected leaves, displayed the same morphological traits as the initial isolates ZX6, according to Koch's requirements. This result indicated that ZX6 is the pathogen leading to leaf spotting disease on Alisma. Colonies of ZX6 were initially white and fluffy on PDA, and gradually turned salmon pink, with few aerial hyphae and slimy surfaces. Conidia were smooth, with 0-1 septa, elliptical or ovoid, measuring 12.2 ~ 19.4 × 2.2 ~ 3.4 µm (average 15.5 × 2.7 µm, n=100). These morphological characteristics were similar to Plectosphaerella spp. (Carlucci et al. 2012; Pitt et al. 2005). For species identification, we extracted mycelium genomic DNA and performed amplification of the internal transcribed spacer (ITS), large subunit of rDNA (LSU), and calmodulin (CaM) genes using primer pairs ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Hopple et al. 1999), and CL1/CL2 (Weir et al. 2012), respectively. In a BLAST search, the ITS, LSU, and CaM sequences showed 99.39% (492 / 495 bp), 100% (850 / 850 bp), and 100% (479 / 479 bp) similarity to Plectosphaerella alismatis strain CBS 113362 in the NCBI database (accession numbers JF780523, KY662261, and KY416925, respectively). Based on the combined ITS, LSU, and CaM sequences, a maximum likelihood phylogenetic tree was constructed using MEGA 11.0. By combining morphological characteristics and phylogenetic analysis, the isolate ZX6 was confirmed as P. alismatis. Our study is the first report describing P. alismatis infection of Alisma plantago-aquatica Linn. leaves in China. This result will contribute to developing effective measures to control the leaf spotting disease of Alisma.