First Report of Epicoccum tobaicum Associated with Leaf Spot on Flowering Cherry in South Korea

Abstract

Flowering cherry (FC, Prunus x yedoensis Matsumura; Somei-yoshino cherry) is an ornamental tree, planted across South Korea and producing stunning flowers in spring. The seasonal blooms are annually celebrated during cherry blossom festivals in many locations across the country. The leaf spot disease is among the most common and important diseases affecting FC trees every year, resulting in premature defoliation and reduced flowering of cherry blossoms in the following year. In May 2018, brown spots (2 to 5 mm), circular to irregular and with dark borders were observed on FC leaves in Hadong, Gyeongsangnamdo, South Korea (35°07'48.9"N, 127°46'53.8"E), with a disease incidence of 55%. Single lesions often coalesced and were sometimes perforated, leaving shot holes. Sampled leaves were surface sterilized with 1% NaOCl for 1 min and 70% ethanol for 30 s, and then rinsed twice with sterile distilled water. About 2-mm-long infected leaf pieces from the margins of lesions were put onto water agar (WA, 1.5% agar) plates and incubated at 25oC for 72 h. Mycelia grown from symptomatic tissue were transferred to PDA plates, and five similar fungal isolates were obtained from hyphal tips. They produced a strong reddish-orange diffusible pigment on PDA after 5 d and exudates after 8 d. Conidia were globular to pear-shaped, dark, verrucose, multicellular, and 14.8 to 23.5 μm in diameter (av. = 18.7 μm, n = 30) for isolate JCK-CSHF10. These morphological characteristics were consistent with the Epicoccum genus. Three loci, ITS, tub2, and rpb2, from three isolates JCK-CSHF8, JCK-CSHF9, and JCK-CSHF10 were amplified using the primer pairs ITS1F/LR5 (Gardes and Bruns 1993; Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/RPB2-7cR (Liu et al. 1999; Sung et al. 2007), respectively. The ITS, tub2, and rpb2 sequences of the three isolates were deposited in Genbank (MW368668-MW368670, MW392083-MW392085, and MW392086-MW392088, respectively), showing 99.6 to 100% identity to E. layuense (E33), a later synonym for E. tobaicum (Hou et al. 2020). The phylogenetic tree using concatenated sequences of the three loci placed the three isolates in a cluster of E. tobaicum (CBS 232.59, CGMCC 3.18362, and CBS 384.36; Hou et al. 2020). Taken together, the three isolates were identified as E. tobaicum. The pathogenicity of JCK-CSHF10 was tested on 15 healthy leaves on three FC trees (cv. Somei-yoshino, 1.2 m in height) kept in a greenhouse. Five-mm-diameter plugs from 7-d-old fungal cultures grown on PDA or mycelia-free PDA plugs as controls were placed on the abaxial side of a leaf at three points, previously wounded by a sterile needle (Zlatković et al. 2016). Inoculation sites were covered with moist cotton plugs. Trees were then covered with a clear plastic bag and maintained in high humidity at 25oC in darkness for 24 h, followed by a 12-h photoperiod. Brown spots appeared on inoculated leaves after 7 d, identical to those observed in the field, while control leaves remained symptomless. This experiment was repeated three times. A fungus with the same morphology as JCK-CSHF10 was recovered from lesions, thus confirming Koch’s postulates. E. layuense (syn. E. tobaicum) has been reported as a leaf spot-causing agent on Perilla sp. (Chen et al. 2017) and Camellia sinensis (Chen et al. 2020). To date, there is no report on the occurrence of E. tobaicum from leaf spots on FC. To our knowledge, this is the first report of E. tobaicum causing leaf spot on FC in South Korea.

Didymellaceae species associated with tea plant (Camellia sinensis) in China

“…It was resurrected as a separate species, distant from E. nigrum ( Hou et al 2020b ). Conidia were globular to pear-shaped, dark, verrucose and multicellular ( Han et al 2021 ). It formed a distinct lineage closely related to E. rosae (Fig.…”

“…3 ). This species as a pathogen was isolated from diseased leaves showing leaf spot of flowering cherry and oat ( Han et al 2021 ; Jeong et al 2022a ). In the present study, five strains were isolated from diseased tea plant leaves.…”

Identification, Characterization, and Efficacy Evaluation of Bacillus velezensis for Shot-Hole Disease Biocontrol in Flowering Cherry

“…Xap P1, the causative agent of leaf spot on peach plants, and Mc, the leaf spot-causing agent of oriental cherry plants ( P. serrulata var. spontanea ), and the three pathogens inducing leaf spots and shot holes on FC (Et JCK-CSHF10, Bc JCK-CSHB12, and Pss JCK-CSHB44) ( Han et al, 2021a , 2021b ) were used. Mc and Et were cultured on potato dextrose agar/broth (PDA/PDB) (Becton, Dickinson, and Co., Heidelberg, Germany) (Becton, Dickinson, and Co., Heidelberg, Germany) at 25°C; Xap, Bc, and Pss were cultured on tryptic soy agar/broth (TSA/TSB) (Becton, Dickinson, and Co.) at 28°C.…”

“…syringae (Pss), and Xanthomonas arboricola pv. pruni (Xap) ( Han et al, 2021a ), and the fungi Mycosphaerella cerasella (Mc) and Epicoccum tobaicum (Et) ( Han et al, 2021b ).…”

Leaf disease caused by Epicoccum thailandicum on Amomum villosum in China

“…are capable of acting as pathogen for plant diseases of tea [Camellia sinensis (L.) Kuntze] leaves (Yin et al, 2021), Weigela florida (Bunge) A. DC. (Tian et al, 2021), and flowering cherry (Prunus × yedoensis Matsumura; Somei-yoshino cherry) (Han et al, 2021).…”