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

<i>Euonymus japonicus</i>, an evergreen shrub, is widely planted in parks and along roadsides in temperate and subtropical regions due to its high adaptability. In January 2025, <i>E. japonicus</i> leaf spot was observed at Weifang University of Science and Technology, Shandong Province, China (118.78°E, 36.89°N), with 20-40% of leaves per plant affected (n = 230 plants). Irregular chlorotic spots appeared on the surface of the leaves, then larger necrotic spots showed grayish-white centers and brown margins formed. Numerous pycnidia appeared at later stages of development. To isolate the pathogen, 20 symptomatic leaves were collected from ten trees and cut into small pieces (0.4 cm²) at the junction between diseased and healthy tissue. All pieces were surface disinfected with 75% ethanol for 30 s then rinsed three times with sterilized distilled water, dried with sterilized filter paper, placed on potato dextrose (PDA), and incubated in darkness at 27 ° C. Seventeen isolates were obtained using the hyphal-tip method, all of which exhibited similar morphological characteristics. Colonies initially appeared white, and after 5-7 days, they gradually turned gray-green to dark gray with dense aerial mycelia. Conidia forming on pycnidia were one-celled, hyaline, fusiform, subtruncate at the base, subobtuse at the apex, and measured 19.4 -25.9 μm (23.1 ± 1.6 μm) × 5.2 - 9.2 μm (7.1 ± 0.9 μm) (n = 30), consistent with the description of <i>Botryosphaeria dothidea</i> (Zhang et al. 2021). Genomic DNA was extracted from the mycelia of 6-day-old cultures of two representative isolates, DQWM01 and DQWM02. PCR amplifications were performed using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, and Bt2a/Bt2b (White et al. 1990; Carbone and Kohn 1999; Jia et al. 2019) to target the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (<i>TEF1-α</i>) gene, and β-tubulin (<i>TUB2</i>) gene, respectively. Sequences (GenBank accession nos. PV413678 and PV413679 for ITS, PV441490 and PV441491 for <i>TEF1-α</i>, PV441488 and PV441489 for <i>TUB2</i>) showed 99.5 to 100% homology with ITS (AY236949), <i>TUB2</i> (AY236927) and <i>TEF1-α</i> (AY236898) sequences from ex-type <i>B. dothidea</i> isolate CBS 115476. Results from the maximum likelihood analyses, based on the three concatenated gene sequences using PhyloSuite version 1.2.2 (Zhang et al., 2020), placed the isolates in a clade with <i>B. dothidea</i>. To assess pathogenicity, ten healthy leaves from each of five 1-year-old <i>E. japonicus</i> plants grown in a greenhouse were wounded with a sterilized needle. Mycelial plugs (6 mm diameter) from actively growing colonies of isolate DQWM01 on PDA were applied to 5 leaves of <i>E. japonicus</i>, while the other 5 wounded leaves on the same plant were treated with agar plugs. All plants were incubated at 25°C, 70% humidity, and 14 h/10 h photoperiod. Seven days after inoculation, brown lesions appeared, which turned grayish-white with brown margins, similar to field symptoms, but the control leaves remained symptomless. <i>B. dothidea</i> culture was reisolated from all the infected leaves and identified using morphological characteristics and DNA sequence analysis, completing Koch's postulates. The pathogenicity test was performed three times with similar results. To our knowledge, this is the first report of <i>B. dothidea</i> in China causing leaf spot on <i>E. japonicus</i>. The disease significantly threatens <i>E. japonicus</i>'s ornamental value, requiring urgent and targeted control measures.