First Report of leaf spot disease on <i>Codonopsis pilosula</i> (Franch.) Nannf. Caused by <i>Albifimbria verrucaria</i> in Shanxi, China

Codonopsis pilosula (Franch.) Nannf, a medicinal plant in Campanulaceae family, is rich in bioactive compounds such as polysaccharides, saponins, and flavonoids, exhibiting immunomodulatory, antioxidant, anti-fatigue, and organ-protective activities (Shi et al. 2024). In August 2024, leaf spot symptoms were observed on approximately 30% of C. pilosula plants in a cultivation field in Pingshun County, Changzhi City, Shanxi Province, China (36°06′23″N, 113°24′03″E). Symptomatic leaves were collected for further study. Initial symptoms appeared as small, nearly circular lesions on the leaves, with a brown necrotic center surrounded by a yellow halo. These lesions later developed into irregular brown to black spots with distinct margins and slight leaf shrinkage. On severely affected leaves, multiple lesions coalesced, resulting in leaf withering and abscission. To isolate the pathogen, symptomatic tissues were surface-sterilized, rinsed, and placed onto potato dextrose agar (PDA) plates. Cultures were incubated at 25 °C for 5–7 days under a 12 h light/12 h dark photoperiod. Forty-three monosporic isolates were obtained and cultured on PDA. Isolate AD3-2 was selected as the representative strain. Colonies on PDA were circular and flat with smooth, regular margins and an opaque surface. Aerial mycelia were white and floccose at the early stage, later forming concentric rings with sporodochia scattered on aging mycelia, with color gradually changing from pale yellow to dark green or black. Conidiophores were fasciculate and stout, unbranched in the lower portion but broom-like branched in the upper portion, bearing whorls of 3-7 phialides at their apices. Conidia were borne singly at the tips of phialides, ellipsoidal with slightly tapering ends, measuring on average 7.95 (6.9–9.0) μm × 2.1 (1.6–2.6) μm (n = 100). Based on its morphological characteristics, the pathogen was identified as Albifimbria verrucaria (Lombard et al., 2016). To confirm identification, partial sequences of the internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), and calmodulin (CAL) gene regions were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), fRPB2-5F2/fRPB2-7cR (Sung et al. 2007), and CAL-228F/CAL2Rd (Carbone &amp; Kohn, 1999), respectively. The sequences were deposited in DDBJ ITS: LC903080; RPB2: LC903081; CAL: LC903082. BLAST analysis revealed that the ITS and RPB2 sequences of isolate AD3-2 shared 100% identity with those of the A. verrucaria (syn. Myrothecium verrucaria) type strain CBS 328.52, and the CAL sequence shared 99.15% identity (Lombard et al., 2016), supporting its identification as A. verrucaria. Phylogenetic analysis of a concatenated ITS, CAL, and RPB2 dataset using neighbor-joining and maximum likelihood methods in MEGA X (Kumar et al., 2018) showed that isolate AD3-2 clustered with the typical strain of A. verrucaria. To confirm its pathogenicity, the isolate was inoculated onto healthy potted C. pilosula plants by applying three 10-μL drops of a conidial suspension (1 × 10⁶ conidia/mL) to each leaf, while control plants were mock-inoculated with sterile distilled water. Three plants per treatment and three leaves per plant were used, giving nine inoculated leaves per treatment. After inoculation, plants were placed in an inoculation chamber and covered with plastic bags to maintain a relative humidity of 70–80%. On the fifth day after inoculation, typical leaf spot symptoms developed on all inoculated leaves, and the lesions were identical to those observed in the field, whereas no symptoms developed on control plants. Reisolation of the fungus from the inoculated leaves, showing morphological characteristics and sequences consistent with isolate AD3-2, confirmed that the causal agent was A. verrucaria, thus fulfilling Koch’s postulates. This study confirmed that A. verrucaria is the causal agent of leaf spot disease on C. pilosula (Franch.) Nannf., representing the first report of this disease caused by A. verrucaria under natural conditions in China. Further research is needed to elucidate the epidemiology and transmission of this pathogen and to develop effective management strategies for disease control.