First Report of Paramyrothecium vignicola Causing Leaf Spot on Forsythia suspensa in China.

Abstract

Forsythia suspensa, a medicinal plant with significant pharmacological and economic value in China (Wang and Ren 2014), is widely cultivated for its therapeutic applications. In September 2023, leaf spot symptoms were observed on one-year-old F. suspensa plants in a 0.267 ha F. suspensa plantation in Chadian Town, Yunyang District, Shiyan City, Hubei Province, China (32°46'6″N, 110°49'59″E). The plants were locally sourced from Shiyan City and propagated via seeds. Systematic sampling of 50 plants across 10 randomly selected quadrats (5 × 5 m) revealed a disease incidence of 20-30%. Symptoms included circular to irregular brown necrotic lesions (4-10 mm in diameter) with dark borders and prominent concentric rings. Notably, small dark sporodochia were observed within lesions. No canker, stem lesion, or plant mortality was observed during the survey. To isolate the pathogen, 20 symptomatic leaf samples were randomly collected from 10 infected F. suspensa plants. Diseased tissues (5 × 5 mm) excised from lesion margins were surface-sterilized with 75% ethanol for 30 s, followed by 1.5% sodium hypochlorite for 1 min, and rinsed three times with sterile distilled water. The tissues were transferred to potato dextrose agar (PDA) plates and incubated at 28°C. After 10 days, 15 morphologically similar fungal isolates L1-L15 were obtained. Colonies initially exhibited white aerial mycelia, later developing dark green to black conidial masses arranged in concentric rings with sporodochia. Microscopic analysis revealed conidiogenous cells phialidic, cylindrical to subcylindrical, hyaline, smooth, straight to slightly curved (12.03 to 15.93 × 2.01 to 2.47 μm; n=30). Conidia aseptate, hyaline, smooth, cylindrical to eliosoidal (4.95 to 6.96 × 1.07 to 2.56 μm; n=30) with rounded ends, setae arising from the stroma. These morphological characteristics were consistent with the description of Paramyrothecium sp. (Lombard et al. 2016; Withee et al. 2022). Genomic DNA of isolate L2 was extracted from fresh mycelium using DNA extraction kit (TSP001, Tsingke, Co., Ltd). The internal transcribed spacer (ITS) and calmodulin (cmdA) gene regions were amplified with primers ITS4/ITS5 (White et al. 1990) and CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al. 2013), respectively. BLASTn analysis indicated that the ITS sequence of isolate L2 (GenBank accession PP379914; 610 bp) displayed 100% identity with the type Paramyrothecium vignicola isolate SDBR-CMU376 (GenBank accession MZ373242; 601 bp). The cmdA sequence of isolate L2 (GenBank accession PP382839; 684 bp) exhibited 99.42% identity to P. vignicola isolate SDBR-CMU374 (GenBank accession OM810410; 942 bp). Results of double-loci phylogenetic tree analyses based on ITS and cmdA showed isolate L2 clustered within the same branch as P. vignicola. Therefore, based on morphological characteristics and molecular phylogeny, isolate L2 was identified as P. vignicola. To evaluate pathogenicity, three healthy leaves of each potted one-year-old F. suspensa plant were sprayed with conidial suspensions (1×106 conidia/mL). In addition, the left sides of leaves were wounded by pricking with a sterile needle. Three plants were treated in the same way and sprayed with sterile distilled water as control. Each treatment was repeated three times. All inoculated plants were covered to maintain high humidity and placed in a moist chamber at 25°C, 95% relative humidity. After eight days, brown lesions identical to field symptoms developed on inoculated leaves (wounded), whereas the control leaves remained free of symptoms. P. vignicola was reisolated only from randomly selected symptomatic leaves, fulfilling Koch's postulates. P. vignicola has been reported as a new species infecting Solanum virginianum, Lablab purpureus, Coccinia grandis and Brassica chinensis in Thailand (Haituk et al. 2024; Withee et al. 2022). However, to our knowledge, this is the first report of P. vignicola infecting F. suspensa in China. The identification of the pathogen provides a theoretical basis for the prevention and control of this disease.