LAUSR

American ginseng (Panax quinquefolius L.) is an herbaceous perennial understory plant. It was listed as endangered species by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (McGraw et al. 2013). Leaf spot symptoms were observed on 6-year-old cultivated American ginseng on a research plot (8 x 12 ft raised bed under a tree canopy) in Rutherford Co., TN in July 2021 (Fig. 1a). Symptomatic leaves were exhibiting light brown leaf spots with chlorotic haloes 0.5 to 0.8 cm in diameter, mostly confined within or bounded by veins. As the disease progressed, leaf spots expanded and coalesced into irregular shapes with necrotic centers, resulting in a tattered appearance of the leaf. Disease severity was about 50 to 80% of leaf area and incidence was 10% out of 20 plants. Plant tissues were surface sterilized with 10% NaOCl2 for 60s and washed thrice with sterile water and plated on potato dextrose agar (PDA). Colony growth of the isolates FBG880 and FBG881 on PDA were round, white, thick, and flocculent at the front of the plate and showed a yellowish-ringed shape on the back 10 days after incubation at 25°C (light/dark: 12/12h). Acervular conidiomata containing abundant conidia were observed on PDA. They were globose, 1.0 to 1.8 mm in diameter, and found as solitary or aggregated clusters. Conidia contained five cells (average 13.03±3.50 x 14.31±3.93 µm, n = 30). The middle three cells were light brown to brown. The basal and apical cells were nearly triangular, and transparent, with two to three (7:3 ratios, respectively) apical appendages (average 13.27±3.27 µm) and a basal appendage (average 4.50±0.95 µm, n = 30). To determine pathogen identity, total DNA was extracted using DNeasy PowerLyzer Microbial Kit from fungal colonies on PDA (isolates FBG880 and FBG881). The ribosomal internal transcribed spacer (ITS) region, beta-tubulin (BT), and translation elongation factors 1-α (EF1) genetic markers were amplified using ITS1/ITS4 (White et al. 1990), T1/T2 (Stefańczyk et al. 2016), and EF1/EF2 (O'Donnell et al. 1998), respectively. The sequences (GenBank accession nos. ITS: OQ102470 and OQ103415; BT: OQ107059 and OQ107061; and EF1: OQ107060 and OQ107062) are 100% similar to Pestalotiopsis nanjingensis (CSUFTCC16 and CFCC53882) (Jiang et al. 2022; Li et al. 2021) (Fig. 2). Based on morphology and molecular characteristics, the isolates were identified as P. nanjingensis. To conduct the pathogenicity trial, six healthy 1-year-old American ginseng plants, germinated from seeds and grown in the greenhouse were spray inoculated with a conidial suspension (1×106 conidia/ml) (FBG880). Six control plants were sprayed with sterile water. All plants were covered with plastic bags and incubated in a greenhouse set at 21 to 23°C, 70% relative humidity and 16-h photoperiod. After 48 h, bags were removed and plants were maintained under the same conditions. After one month, while control plants remained asymptomatic (Fig. 1b), inoculated plants started to exhibit symptoms resembling those in the research plot (Fig. 1c). Fungal isolates resembling P. nanjingensis in cultural characters were consistently recovered from inoculated plants and their identity as P. nanjingensis was confirmed by DNA sequencing. To our knowledge, this is the first report of leaf spot disease caused by P. nanjingensis on American ginseng. Identification of this pathogen and confirmation of its pathogenicity are fundamental to future disease management approaches.