The genus Cercospora Fresen. ex Fuckel (Mycosphaerellaceae) contains numerous economically and ecologically important plant pathogenic fungi that cause leaf spot disease on various plants, including angiosperms, gymnosperms, and ferns [1–5]. The current concept of the genus was established in 2013, proposing the use of multilocus sequence analysis for species delimitation within the genus [4]. Since then, several protein-coding genes, such as actin (actA), β-tubulin (tub2), calmodulin (cmdA), glyceraldehyde-3phosphate dehydrogenase (gapdh), histone H3 (his3), translation elongation factor 1-alpha (tef1), and RNA polymerase II second largest subunit (rpb2), along with the internal transcribed spacer (ITS) region of the rDNA, have been investigated [6].

Among the poorly known and fastidious species of Cercospora and cercosporoid fungi, C. flagellaris Ellis & G. Martin remains unresolved, and its taxonomy has become increasingly complex with the emergence of new reports globally. This fungus was first described in 1882 as a leaf spot pathogen of Phytolacca decandra L. (currently Phytolacca americana L.) in North America, which is the native range of the host [7]. Numerous isolates that phylogenetically cluster within the flagellaris group have been designated as confer (cf.) flagellaris owing to several uncertainties. Bakshi et al. [6] noted that the cf. flagellaris group can be divided into three distinct clades based on analyses of eight gene loci in molecular phylogenetics. However, this remains insufficient for species delimitation because of overlaps in morphology and host ranges among these clades within the group. Currently, the number of C. cf. flagellaris isolates from various hosts continues to expand, encompassing plants from diverse families [4,8,9]. However, C. f lagellaris s. str. on Phytolacca spp. has been reported in North America and East Asia, with the exception of Ethiopia [10,11]. To date, only three isolates of C. flagellaris have been analyzed using multigene sequence data by Groenewald et al. [4], all of which originated from Korean samples of P. americana.

In Korea, this fungus was first reported on Phytolacca esculenta Van Houtte [12], followed by a report on P. americana [13]. The authors noted abundant hypophyllous fructification of the fungus on some samples that co-occurred with epiphyllous fructification. They compared their findings with Chupp’s description of American specimens, which were characterized by epiphyllous caespituli.

The first step in resolving the complexity of the flagellaris group was to elucidate the morphology and molecular phylogeny of true C. flagellaris on its original host. Therefore, this study aimed to reassess and integrate previous records with newly obtained collections of C. flagellaris on P. americana in Korea. By combining morphological examinations with multigene phylogenetic analyses, this study sought to clarify the identity of true C. flagellaris on its original host.

The samples used in this study are listed in Table 1, along with their collection dates, localities, strains, and sequence deposition IDs in the relevant databases. All collected samples were preserved at the Herbaria of Korea University (labeled as KUS-F, Seoul, Korea) and Jeonbuk National University (as JBNU-F, Jeonju, Korea). For morphological characterization of C. flagellaris, fungal structures were examined and photographed using a Zeiss AX10 microscope equipped with an AxioCam MRc5 camera (Carl Zeiss, Oberkochen, Germany). The size of each diagnostic feature was determined based on at least 20–30 measurements. To obtain single-spore isolates, conidia collected from young lesions were mounted on a drop of sterile water and streaked onto 2% water agar (WA; Junsei, Tokyo, Japan) plates supplemented with 100 mg/L streptomycin sulfate. The plates were incubated at 25°C. After 2 days, the germinating conidia were transferred to 2% potato dextrose agar (PDA; Difco, Detroit, MI, USA) plates to obtain a pure culture. Colony color was determined using Raynar color charts [14]. The obtained strains were deposited in the Korean Agricultural Culture Collection (KACC; Wanju, Korea).

All examined specimens were consistent with the leaf symptoms and fungal morphology. Initial lesions on the affected leaves were circular to irregular, brown to pale brown, and more or less sunken in the middle. As the spots enlarged with narrow borders, they became tan, creamy white, and even grayish with heavy fructification. Discrete lesions were mainly less than 6 mm in diameter, occasionally confluent, and cottony with numerous conidiophores and conidia (Fig. 1A, B). Stromata were small to medium, poorly developed, sometimes only occupying stomatal openings, brown to dark brown, 10–24 μm in diameter, and composed of several dark brown cells (Fig. 1C, D). Conidiophores were 8–16 in a divergent fascicle, emerging through stomatal openings in the early stage or from the cuticle of the upper leaf surface in the later stages of disease development. They were usually not branched, mildly or abruptly geniculated 1–4 times, olivaceous brown throughout or paler upwards, variable in length, 30–265 × 3–5 μm, and 0–5-septate (Fig. 1E). Conidial scars on conidiophores were conspicuous, apical, or at the position of geniculations, and 3–5 μm in diameter. Conidiogenesis was holoblastic, with terminal conidiogenous cells. Conidia were solitary, acicular-filiform, straight to mildly curved, hyaline, 4–12-septate, 40–160 × 3.0–4.5 μm, with conspicuously thickened and darkened hila, base truncate to subtruncate, and apex obtuse (Fig. 1F).