First Report of <i>Botryosphaeria dothidea</i> Causing Leaf Spot on <i>Euonymus fortunei</i> in China

<i>Euonymus fortunei</i> (Turcz.) Hand.-Maz., an evergreen vine shrub, commonly used for landscaping and herbaceous medicines in China. In December 2024, leaf spot symptoms were noticed on <i>E. fortunei</i> plants in three parks with a 15-25% disease incidence out of 160 trees in Weifang City, Shandong Province in China (118°44'34.9''E, 36°52'17.3''N). Irregularly shaped lesions with a grayish-white center and brownish margins surrounded by a yellow halo developed on infected leaves and coalesced at later stages. To isolate the causal agent, 14 diseased leaves were randomly collected from 10 trees in different parks and a small piece (2 x 2 mm) was sliced from each lesion at the edge, surface-disinfected with 75% ethanol for 30 s, rinsed 3 times in sterilized water, placed on a potato dextrose agar (PDA), and incubated at 27°C in the dark for 3 days. <i>Botryosphaeria</i> sp.-like fungal colonies were isolated from 93% of the sampling plates and 3 representative fungal isolates (FFT01 to FFT03) were selected randomly for morphological and molecular characterization and hyphal-tip purified. Fungal colonies initially appeared to be white with fluffy mycelia and gradually developed a grayish-black pigment. The hyaline, unicellular, and oblong-fusiform conidia were 20.1 - 25.7 μm (mean = 23.2 ± 1.4) long and 6.3 - 9.1 μm (mean = 7.2 ± 0.8) in diameter (n = 30), similar to the morphological features of <i>Botryosphaeria dothidea</i> (Zhang et al. 2021). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (<i>tef1</i>) gene, and beta-tubulin (<i>tub2</i>) gene were amplified and sequenced using primer pairs ITS1/ITS4, EF1- 728F/EF1-986R, and Bt2a/Bt2b, respectively (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999) and desposited in GenBank (Accession Nos. PV422744 to PV422746 for ITS, PV441492 to PV441494 for tef1, and PV4414485 to PV441487 for tub2). Sequences had > 99% identity with their corresponding sequences from the ex-type <i>B. dothidea</i> strain CMW 8000. In the maximum-likelihood phylogeny derived from the concatenated alignments, all three isolates were grouped in the <i>B. dothidea</i> clade. To validate the pathogenicity of FFT01 isolate, five leaves from each of three one-year-old <i>E. fortunei</i> seedlings were scratched with a sterilized syringe needle and then a 5 mm² mycelial PDA plug of 6-day-old culture was placed on each wounded site. Three <i>E. fortunei</i> seedlings inoculated with sterile PDA plugs served as controls. Each wound-inoculated seedling was enclosed in a transparent plastic bag for 3 days then kept in a growth chamber under controlled conditions (27°C, 14/10-h light/dark cycle, and 75% relative humidity). Seven days post-inoculation, grayish-white lesions surrounded by yellow halo were observed on leaves with black pycnidia developing in lesions, similar to the symptoms on the naturally infected leaves, while no lesions appeared on mock-inoculated leaves. The identity of the fungus re-isolated from symptomatic leaves was confirmed as <i>B. dothidea</i> based on morphology and DNA sequence. The pathogenicity test was repeated three times with similar results. Although <i>B. dothidea</i> has been reported as a common fungal pathogen causing leaf spot, fruit rot or stem canker on a variety of plants including plum, <i>Kadsura coccinea</i>, and soybean (Chen et al. 2021; Su et al. 2024; Yuan et al. 2024), this is the first report on its occurrence on <i>E. fortunei</i> in China. This study will provide a basis for the prevention and control of leaf spot on <i>E. fortunei</i> in the future.