First Report of Leaf Spot on Cinnamomum migao Caused by Epicoccum latusicollum in China

Cinnamomum migao, a plant in the Lauraceae, is a rare and endangered species endemic to China (Yan et al. 2022). In February 2025, symptoms of leaf spot were observed on C. migao plants growing in Pingtang County, Guizhou Province, China (25°29'55″N, 106°40'29″E). Disease incidence reached 100% (n=200). Foliage exhibited ash-white necrotic lesions that were elliptical or irregular in shape, surrounded by a brown halo at the margin. Six symptomatic leaf samples (5 × 5 mm) were surface-sterilized with 75% ethanol for 30 s, followed by 2.5% NaClO for 1 min. After air-drying, the tissues were placed on potato dextrose agar (PDA) and incubated at 28°C for 7 days. Hyphal tips from emerging colonies were transferred to fresh PDA plates to obtain pure cultures. A total of 16 purified isolates were obtained, 12 of which shared similar morphological characteristics. One representative isolate (designated ZYX) was selected for further study. Colonies on PDA were villous, with dense aerial mycelia, initially white and turning grayish over time; the reverse side was white with a yellow gradient and a red concentric ring. Conidia were ellipsoidal, aseptate, and 3.7 - 6.4 x 1.4 - 4.0 μm (average 5.04 × 2.61 μm; n= 60). Morphological characteristics were consistent with those described for Epicoccum latusicollum, a fungal pathogen causing leaf spot on tobacco in China (Guo et al. 2021). For molecular identification, the internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 (White et al. 1990), the large subunit ribosomal RNA gene (LSU) with LROR/LR5 (Vilgalys and Hester 1990), the beta-tubulin gene (TUB2) with Btb2Fd/Btub4Rd (Woudenberg et al. 2009), and the RNA polymerase II second largest subunit gene (RPB2) with RPB2-5F2/fRPB2-7cR (Liu et al. 1999). Resulting sequences were deposited in GenBank with accession numbers PV946034 (ITS), PV946035 (LSU), PV976759 (TUB2), and PV976758 (RPB2). BLASTn analysis showed isolate ZYX shared 99% to 100% sequence identity with the E. latusicollum type strain CGMCC 3.18346, ITS (KY742101; 478/479 bp), LSU (KY742255, 859/863 bp), TUB2 (KY742343, 333/333 bp), and RPB2 (KY742174, 595/596 bp) sequences in the GenBank database. Furthermore, the amplified sequences were used to construct a phylogenetic tree in PhyloSuite 1.2.3 which showed that ZYX clustered with E. latusicollum. Thus, both morphological and molecular data support the identification of the isolate as E. latusicollum. To fulfill Koch's postulates, pathogenicity tests were conducted using conidial suspensions (1×106 conidia/mL) sprayed onto three healthy C. migao plants. Three additional plants were treated with sterile distilled water as controls, and plants were maintained at 28°C with 85% relative humidity and a 12-hour photoperiod. After 7 days, the inoculated plants developed symptoms identical to those observed in the field, while the control plants remained asymptomatic. The pathogen was successfully re-isolated from symptomatic leaves and confirmed based on morphological features and DNA sequences, fulfilling Koch's postulates. No pathogens were isolated from the control plants. E. latusicollum has been reported to cause disease in various medicinal plants (Yan et al. 2024, Wang et al. 2023). To our knowledge, this is the first report of E. latusicollum causing leaf spot on C. migao in China. This report contributes to the development of targeted and efficient disease management strategies for growers.