First Report of Leaf Spot Disease Caused by <i>Corynespora cassiicola</i> on <i>Rhododendron pulchrum</i> in China

The significant species Rhododendron pulchrum Sweet, prized for its exceptional ornamental characteristics, has been widely cultivated across eastern China's major production regions, particularly in Jiangxi, Fujian, and Zhejiang provinces (He et al., 1994). This plant demonstrates significant value as an ornamental potted cultivar for festive occasions, well-suited for cultivation in diverse green areas such as parks, streets, communities, and schools. However, since 2024, severe leaf spot on R. pulchrum with disease incidence of 50 to 80% were observed in about 240 plants in campus green belt located in Jiujiang University (29°68’30.55’’ N, 116°00’33.13’’ E), Jiangxi Province, China. The first signs of infection manifested as small brown spots (2 to 3 mm) along the main vein of the leaves, progressively enlarged into reddish-brown necrotic areas before coalescing into irregular or circular lesions. Seventy-five diseased leaves were collected and used for isolation. The lesion margins were excised into 2 × 2 mm² sections, and the leaf surfaces were sterilized with 70% ethanol for 5 seconds before being immersed in a 0.1% mercury solution for 60 seconds. Subsequently, the fragments underwent triple rinsing in sterile water, followed by placement onto potato dextrose agar (PDA) medium using sterile tweezers, and were then incubated at 25℃. The germinated spores were subsequently transferred to PDA to establish single-spore cultures for morphological examination. Two fungal isolates confirmed as conspecific through combined morphological and molecular characterization were obtained. A sole representative strain, jjtu-GL2501, was selected for further analysis. The colony of this isolate measured about 7.1 cm in diameter after seven days of cultivation on PDA. The side of the colony was light gray in color, and the center was gray or dark brown. Conidia were pale brown, simple, cylindrical, and septate with intermittent branching. The conidia were solitary or 2 to 4 clusters, long cylindrical, straight to slightly curved, pale olivaceous to brown, ranged from 21.2 to 130.3 μm in length and 4.3 to 7.8 μm in width (n = 50). The morphological characteristics matched those of Corynespora cassicola (Zhang et al. 2018). For molecular identification, genomic DNA was extracted from mycelia of two representative isolates (jjtu-GL2501 and jjtu-GL2505) by CTAB method (White et al. 1990). Three regions of the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1-α), and RNA polymerase Ⅱ subunit B (RPB2) genes were amplified and sequenced with the primer pairs ITS5/ITS4 (White et al. 1990), EF1-983F/EF1-1567R (Rehner and Buckley 2005), and RPB2-6F/RPB2-7R (Liu et al. 1999), respectively. Sequences (GenBank accession nos. PV202844 and PX426502 for ITS, PV847685 and PX677440 for TEF1-α, PV847686 and PX677439 for RPB2) showed 99.56 to 100% homology with ITS (MT919867), TEF1-α (OM892998) and RPB2 (OM937067) sequences from C. cassiicola. Based on the concatenated ITS, TEF1-α and RPB2 sequences, a maximum-likelihood phylogenetic analysis using MEGA7.0 clustered the two isolates (jjtu-GL2501 and jjtu-GL2505) with reference C. cassicola. Based on morphological and molecular analysis, two isolates were identified as C. cassicola. To fulfill Koch's postulates, a conidial suspension (1 × 10⁶ conidia/ml) derived from isolate jjtu-GL2501 cultured on PDA at 25°C for 7 d was used to inoculate both mechanically wounded and intact leaves of potted seedlings. Fresh wounds were created with a sterile needle, while control leaves received sterile water treatments. The experiment employed three replicates per treatment with two independent trials. Inoculated plants were immediately transferred to a growth chamber maintained at 25°C. The leaves were observed every day, and symptoms were observed, counted and photographed. Distinct disease symptoms consistent with field observations manifested on jjtu-GL2501-inoculated plants at 5 days post-inoculation, whereas control plants remained asymptomatic. The fungus was successfully reisolated from the symptomatic leaves and conclusively identified as C. cassicola through combined morphological characterization and multi-gene sequence analysis. The validation of Koch's rule was completed. To our knowledge, this is the first reported of C. cassiicola on R. pulchrum in China. This finding helps identify the pathogen accurately and provides a scientific basis for the development and implementation effective disease prevention and control strategies.