First Report of Leaf Blight Caused by <i>Botryosphaeria dothidea</i> on <i>Idesia polycarpa</i> in China

The wonder tree (Idesia polycarpa), a deciduous species in the Salicaceae family is widely distributed in China and serves both ornamental and economic purposes. In May 2024, a blight symptom was observed on approximate 60% of leaves of wonder trees located at Weifang University of Science and Technology (36°53′42.16″N, 118°47′22.4″E). Initially small brown spots appeared on the leaves, then expanded to large irregular grayish-white lesions with dark-brown edges. Ten symptomatic leaves were sampled from 10 infected trees in a 0.3 ha orchard. Fifteen sample tissues (3 × 3 mm) were sterilized with 1% sodium hypochlorite for 10 s followed by 75% ethanol for 30 s, rinsed twice in sterile distilled water and placed on a potato dextrose agar (PDA) plate. The plates were incubated at 25°C for 3 days in darkness. Ten isolates were obtained with similar morphological characteristics using the hyphal-tip method. Three isolates (S2, S5, S6) were used for the morphological characterization and molecular analysis. The colonies reached 8.5 cm in diameter on the PDA plates after 5 days incubation at 25°C。The colonies were initially white, and gradually turned to dark grey with dense aerial mycelia after five days, while the reverse side of the colonies appeared gray to black. Conidiophores were hyaline, cylindrical and smooth. Conidiogenous cells were holoblastic, hyaline, smooth, cylindrical to ampulliform and produced a single conidium at the tips. Conidia were fusiform, aseptate, straight and hyaline, 22–30 × 5–8 μm (av. of 50 conidia=26.2 × 7.3 μm). The morphological characteristics matched the description of Botryosphaeria dothidea (Crous et al., 2006; Slippers et al., 2014). To further confirm the causal fungus of leaf blight on I. polycarpa, the internal transcribed spacer (ITS)，β-tubulin (TUB2) and elongation factor 1-α (TEF1-α) genes were amplified using ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R primers, respectively (Jia et al. 2019). The sequences (GenBank accession nos. PQ252929 – PQ252931 for ITS; PQ349189 – PQ349191 for TUB2; PQ349192 – PQ349194 for TEF1-α) were aligned in GenBank showing >99% homology with the sequences of ITS (AY236949), TUB2 (AY236927) and TEF1-α (AY236898) from isolate CMW8000. The maximum-likelihood analysis was performed using concatenated sequences of ITS, TUB2 and TEF1-α. The phylogenetic analysis confirmed that all isolates clustered with B. dothidea. A pathogenicity test was performed on 20 healthy leaves of five 8-year-old I. polycarpa trees by a wound inoculation. Mycelial plugs from 5-day-old fungal colonies on PDA were inoculated to ten leaves, and ager plugs were inoculated to the same number of leaves as controls. Plastic bags were used to wrap the leaves with a wat cotton ball inside to maintain moisture conditions. After seven days, all inoculated leaves displayed lesions similar to those observed in the field, whereas controls remained asymptomatic. The pathogenicity tests were repeated three times. The fungus reisolated from the inoculated leaves were further identified as B. dothidea based on morphological characterization and molecular analyses. B. dothidea has also caused leaf blight on ornamental plants, including Mahonia fortunei (Huang et al., 2023), Parrotia subaequalis (Yang et al., 2024) and Syringa oblata (Zhi et al., 2024). This is the first report of B. dothidea causing leaf blight on I. polycarpa in China and provides a foundation for the management of the disease.