Epicoccum layuense Causing Leaf Spot on Oxalis corymbosa in China

Oxalis corymbosa DC. is a perennial herb used as the groundcover in the urban landscape and traditional Chinese medicine. In May 2019, a leaf spot-like disease was observed on about 20% of O. corymbosa plants in one park in Hefei, Anhui, China (N31°52´, E117°15´). Symptoms appeared as yellowish, irregular or circular areas that later turned pale brown, coalesced and produced large necrotic areas of various shapes. Ten leaves with typical symptoms were removed from 5 different O. corymbosa plants (2 leaves per plant) and disinfected with 75% ethanol for 30 s. The tissue pieces (4×4 mm) were cut from the margin of the leaf lesion, dipped in 2% NaClO for 1 minute, washed 3 times with sterile distilled water, placed on Potato Dextrose Agar (PDA) medium with 50 μg/ml each of ampicillin and kanamycin, and cultured at 25℃ under a 12-h light/dark cycle. The white mycelia grew out of each leaf tissue pieces 3 days later and the hyphal tips were sub-cultured into a new PDA medium for purification. After 5 days of incubation at 25℃, the produced colonies were yellow with irregular white margin from the top with floccose aerial mycelia, ranged in a diameter from 60 to 70 mm, and produced red orange pigments into PDA medium. Colonies on the reverse side were dark red. Conidia produced on PDA were ranged in size from 5.5 to 35.5 μm in diameter (n = 800), subglobose to pyriform, and yellowish-brown. The representative isolate YHF0519 was chosen for further analysis. Fragments of four genes including the internal transcribed spacer (ITS), beta-tubulin, 28S large subunit ribosomal RNA gene (LSU), and the second largest subunit of RNA polymerase II (RPB2) were amplified from the extracted genomic DNA of YHF0519 and sequenced. The corresponding primer sets were ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), LSU1Fd/LR5 (Crous et al. 2009; Vilgalys and Hester 1990), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007; Liu et al. 1999), respectively. All resulting sequences were deposited into GenBank and received assigned accession numbers: MN418006 for ITS; MN428043 for beta-tubulin; MN428044 for LSU; MZ437946 for RPB2. A phylogenetic tree generated by the maximum likelihood method with 1,000 bootstrapping replications based on the combined ITS, beta-tubulin, LSU, and RPB2 sequences revealed that YHF0519 was clustered closest to the isolate CGMCC 3.18362 of Epicoccum layuense Qian Chen, Crous & L. Cai (Chen et al. 2017; Jayasiri et al. 2017). The pathogenicity test was performed on leaves of healthy O. corymbosa plants in the field and repeated twice. The average air temperature was about 28°C during the pathogenicity test. A total of 126 and 127 leaves were used for treatment and control, individually. Leaves were inoculated by spraying 30 ml of spore suspension (1×10⁶ spores/ml) of YHF0519 or sterilized distilled water. Eight day-post inoculation, about 92% of the leaves inoculated with spores developed pale brown spots, which are identical to those occurred naturally in the field. No symptoms were detected on control leaves. The fungus was re-isolated from the symptomatic leaf tissues and showed the same morphological and molecular characteristics as YHF0519. E. layuense has been reported on Perilla sp., oat, and tea plant (Chen et al. 2017; Chen et al. 2019; Chen et al. 2020). This is the first report of E. layuense causing leaf spot on O. corymbosa in China. Due to the potential threat on the ornamental and medicinal value of O. corymbosa by E. layuesne, it will be necessary to develop local management strategies against leaf spot on O. corymbosa.