Triadica sebifera (L.) Small (Euphorbiaceae), commonly known as the Chinese tallow tree, is an endemic deciduous species of significant economic importance in China (Gao et al. 2016). In October 2023, leaf spot disease was observed on T. sebifera in the Longquan Mountain planting area (30.56°N, 104.27°E), Sichuan Province. Field surveys were performed across three representative planting plots, where a total of 150 trees were randomly examined (50 trees per plot). The mean disease incidence was approximately 83%. Initial symptoms included brown to dark brown lesions on leaves, progressing from small, oval to irregular spots (1–3 mm in diameter) to coalesced large necrotic patches with distinct margins. To isolate the causal agent of the disease, infected tissues were collected from 10 diseased leaves, with each leaf sampled from an individual tree. These infected tissues were cut into 5 × 5 mm pieces, and 3 such tissue pieces were plated per 90 mm-diameter potato dextrose agar (PDA) plate. The infected tissues were surface-disinfected with 75% ethanol and 3% sodium hypochlorite for 60 s and 30 s, respectively (Chen et al. 2025), and rinsed three times in sterile water. Subsequently, the tissues were blot-dried with autoclaved filter paper and cultured on PDA amended with streptomycin sulfate (50 μg/mL), and incubated at 25 ± 2 ℃ in the dark for 8 days. A hyphal tip was taken from the edge of the emerging fungal colony and transferred onto fresh PDA plates for purification. A total of nine fungal isolates were recovered from these ten diseased leaves, seven of which (WJ-1 to WJ-7) exhibited consistent morphological characteristics and were selected as representative strains. For morphological observation and conidiation induction, the purified strains were cultured on PDA at 25 ± 2 ℃ under a 12 h light/12 h dark photoperiod for 8 days. Their colony diameters ranged from 68.9 to 76.9 mm (n=7). The colonies on PDA initially exhibited white, cottony mycelium with undulate margins and concentric rings. Black conidial masses formed on the mycelium. Conidia were fusoid, slightly curved, five-celled with four septa and three dark brown median cells; a total of 50 mature conidia were measured, with an average size of 25.6 × 8.8 μm and a range of 22.3–28.9 × 7.5–9.7 μm. The apical cells were hyaline and conical, bearing 2-3 tubular appendages, while the basal cells were hyaline, conical, and tuberculate with a single central appendage. These morphological traits aligned with Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). For molecular identification, the genomic DNA of a representative isolate (WJ-1) was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The ITS, TEF1-α, and TUB2 regions were amplified and sequenced. The primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone & Kohn 1999), and Bt2a/Bt2b (Glass & Donaldson 1995) were used to amplify ITS, TEF1-α, and TUB2, respectively. The sequences were concatenated and analyzed together with morphological characteristics to determine the species identity of the isolate (Shu et al. 2020). The sequences were deposited in GenBank (accession nos. PQ222635 ITS, PV686787 TUB2, PV730094 TEF1-α). BLASTn analysis revealed 98 - 100% identity with N. ellipsospora reference strains (PP838333.1, MT044311.1, OK594056.1). In this study, we used Pestalotiopsis arengae as an outgroup to construct a multigene phylogenetic tree based on the maximum likelihood method and OFPT (Zeng et al. 2023) software, and the isolate clustered in the N. ellipsospora clade with a bootstrap support greater than 90%. The fungi isolated from the diseased T. sebifera leaves were confirmed as N. ellipsospora using morphological and three-gene sequence analysis. Subsequently, pathogenicity testing was conducted by inoculating leaves of ten T. sebifera trees under field conditions. Ten healthy leaves per tree were surface-sterilized with 75% ethanol. No wounding was performed before inoculation. A conidial suspension of the WJ-1 isolate (1 × 10⁷ conidia/mL) was sprayed onto the leaves, with sterile water serving as the control. Transparent plastic bags were used to maintain relative humidity. After 15 days (25 ± 2 ℃, 14 h light/10 h dark), disease severity was assessed qualitatively by observing lesion appearance and expansion. Inoculated leaves developed symptoms similar to those observed on naturally diseased plants, whereas the control leaves remained asymptomatic. N. ellipsospora was re-isolated from the artificially infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity tests were repeated three times with similar results, confirming Koch’s postulates. To our knowledge, this is the first report of N. ellipsospora infecting T. sebifera globally. This study provides a reference for future research on T. sebifera leaf spot disease management, based on the identification of its causal agent.
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