Euonymus japonicus Thunb., belonging to the family Celastreace and native to East Asia, is a widely cultivated evergreen ornamental woody plant with important ecological and economic values. In May 2023, serious leaf blight of E. japonicus occurred in the campus green space at Guiyang University, Guizhou Province, China (26°55'85"N, 106°78'04"E). Early symptom appeared as small, circular light brown spots on the edges or tips of the leaves. Then, the spot developed visible necrosis, initially light brown to dark brown halos with clear margins. Subsequently, severely infected leaves appear totally wilt, and significantly decrease their ornamental values. In a 0.07-ha field, the disease incidence reached to 40-55%. To identify the pathogen, ten typical symptomatic E. japonicus leaves were collected. They were initially immersed in 75% ethanol for 3 min, and by sodium hypochlorite (4% NaClO) solution for 45 s, and ultimately rinsed with sterile distilled water (dH2O) five times for not less than 1 min each time, then, placed the leaves on potato dextrose agar (PDA) medium and cultured for 5 days at 25°C in constant temperature incubator. Cultures were purified to yield eight isolates. Early colonies are white and regularly rounded, gradually turning dark brown to black with fluffy mycelium. Conidia were single celled, smooth, black, spherical or ellipsoidal. The conidia size of the representative strain, GY-2 and GY-3, was averagely 12.3-17.3 µm × 10.8-17 µm (n = 50). The conidiogenous cells were monoblastic, hyaline, globose or ampulliform. Morphology-based identification revealed the strain as Nigrospora spp. (Wang et al., 2017). For further confirmation, PCR of GY-2 and GY-3 DNA was performed with the primers ITS1/ITS4 (White et al., 1990), Bt2a-F/Bt2b-R (Glass and Don-aldson 1995), and TEF1-728F/TEF1-986R (Carbone and Kohn 1999). Sequences of the ITS region, TUB and TEF1 genes from the strain GY-2 and GY-3 were deposited in GenBank. (GY-2: OR999377, PP112221 and PP150467; GY-3: PP406871, PP421045 and PP421046, respectively). BLAST analysis showed GY-2 100%, 100%, and 98.36%; GY-3 99.43%, 98.21% and 100% (ITS region, TEF1, and TUB) identity to N. hainanensis sequences (accession numbers. NR_153480.1, KY019415.1, and KY019464.1; KX986094.1, OP611475.1, and KY019597.1). Additionally, tandem sequences of ITS, TUB and TEF1 constructed by MEGA 7.0 confimed the homology through the phylogenetic tree. Pathogenicity tests were conducted on healthy plants grown, each 5 mm diameter of active growing mycelium plug of isolate GY-2 was attached to 15 leaves from five healthy 2-year-old E. japonicu plants. The same number of leaves in the control group were treated with non-inoculated plugs only. All the plants were incubated at 25°C and 75% relative humidity with a 16-h/8-h photoperiod. After 10 days, no symptoms appeared on the leaves of the control group. In contrast, symptomatic blight appeared on all leaves inoculated with GY-2. Pathogenicity tests were performed five times. Pure strains were re-isolated from diseased leaves and, confirmed to be N. hainanensis based on the above methods. Recently, Nigrospora oryzae was reported as causal agent of leaf spots on Euonymus japonicus in China (Xu et al., 2023). To our knowledge, this study is the first report of N. hainanensis causing leaf blight on E. japonicu. Identification of the etiological agent may provide assistance for sustainable management in the future.
Keywords: Causal Agent; Euonymus japonicus; Fungi; leaf blight; pathogenicity.