First Report of Corynespora cassiicola Causing Leaf spot of Strobilanthes cusia in China.

Abstract

Strobilanthes cusia (Nees) Kuntze is a vital medicinal and industrial herb, planted extensively in southern China (Hu, et al. 2011.). In July and August of 2021, leaf spot incidence on >60% plants and reduced yields >20% for fresh leaves were observed in S. cusia cultivar 'Malan No.1' across the Shufeng whole Township, Xianyou County, Fujian province. Initial symptoms on leaves were observed as small, dark-brown, spots surrounded by a yellow halo, expanding irregularly or into semicircular spots. As symptoms developed, the spots became dark brown, thin and fragile, forming small holes. In severe cases plants were defoliated. The pathogen was isolated from the margin of 60 symptomatic leaf lesions， surfacesterilized with 75% ethanol for 45 s, rinsed three times with sterile water, air dried, and cultured on PDA at 25°C in the dark. Pure cultures were obtained by single-spore isolation after subculture. Ten representative single-spore isolates (MY-1 to MY-10) from 154 pathogens in 10 sampling points were selected for morphological characterization and identification. After 7 days, mycelial colonies were gray to dark gray with few aerial hyphae. Conidia (32.3 to 132.8 × 5.8 to 8.4 μm, average 81.4 × 6.3 μm, n=50) were pale to brown, erect or curved, solitary or in chains, with 0 to 15 pseudosepta. Based on morphological characteristics, the isolates were preliminarily identified as Corynespora cassiicola. Genomic DNA of isolate MY-2 (randomly selected from 10 isolates as representative) was extracted from mycelia using the Ezup DNA extraction kit (Sangon Biotech Co., Ltd. Shanghai, China). The ITS (internal transcribed spacer) region of rDNA, TEF1-α (translation elongation factor 1 alpha) and TUB2 (beta-tubulin) genes were amplified and sequenced with primers ITS4/ITS5, EF1-728F/EF-986R (Wang et al. 2021) and Bt2a/Bt2b (Glass et al. 1995), respectively. BLASTN sequence analyses of ITS (538 bp), TEF1-α (302 bp) and TUB2 (436 bp) of isolate MY-2 (GenBank accessions OK355515, OM339443, OM339442) showed 100%, 97.6%, 100% identity with C. cassiicola in GenBank (Accession numbers JX908713, MW961421, AB539228). A neighbor-joining phylogenetic analysis based on ITS and TEF1-α sequences using MEGA7 showed that MY-2 clustered in the same clade with C. cassiicola. For pathogenicity tests, five S. cusia plants were inoculated onto the adaxial surface of leaves with mycelial plugs from ten isolates of 8-day-oldcultures on PDA. Five leaves per plant were inoculated, covered with wet cotton, and kept in a controlled greenhouse (26~33 °C, RH 80% ~ 90%). Leaves inoculated with sterile PDA plugs served as a negative control. At 3-5 days post inoculation, all 25 inoculated leaves of each isolate showed leaf spot lesions similar to those observed in the field, and control leaves were symptomless. C. cassiicola was successfully reisolated from the diseased leaves. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. In view of morphology, pathogenicity and sequence results, the isolates were identified as C. cassiicola, a pathogen reported from many important crops (Lu et al. 2021). This is the first report of C. cassiicola as a pathogen in China which poses a potential threat to leaf production and S. cusia processing.