First Report of Nigrospora sphaerica causing leaf spot on Chinese yam ( Dioscorea opposita ) in China

Yam (Dioscorea opposita Thumb.) is an economically important crop with dual food and medicinal uses, and is extensively grown in China, notably in Shandong Province. In 2024, a disease survey was conducted in Heze, Shandong Province, China (35.5346°N, 115.5504°E), during which a leaf spot disease was observed on yam, with disease incidence ranging from 25% to 30% in the affected fields. Initial symptoms appeared as small, circular to subcircular, brown spots with pale yellow halos on the upper leaf surface. With disease progression, the lesions enlarged and developed characteristic grayish-white necrotic centers with dark-brown margins, while the surrounding tissues gradually turned chlorotic, leading to pronounced chlorosis around the lesions. On severely affected leaves, adjacent lesions coalesced into large, irregular necrotic areas (Fig. 1), while severely affected plants exhibited extensive foliar chlorosis, marginal blight, premature senescence, and defoliation under field conditions (Fig. 2). To isolate the causal agent, 60 symptomatic yam leaves were collected from representative diseased plants in the surveyed fields for fungal isolation. Tissue pieces (approximately 5 × 5 mm) excised from the margins of lesions were surface‑disinfested sequentially in 1% NaClO for 30 s and 5% ethanol for 30 s, rinsed three times with sterile distilled water, and dried on sterile filter paper. The sterilized tissues were placed on potato dextrose agar (PDA) plates and incubated at 25°C. After 7 days of incubation, hyphae emerging from the tissues were transferred to fresh PDA plates, and pure cultures were obtained by hyphal tip isolation. Five fungal isolates with similar cultural and morphological characteristics were consistently isolated from symptomatic tissues. One representative isolate, designated YBB-5, was selected for subsequent morphological, molecular, and pathogenicity analyses. On PDA, colonies of isolate YBB-5 reached approximately 80–85 mm in diameter after 7 days at 25°C, appearing white with abundant aerial mycelia. With continued incubation for 10 days, colonies gradually turned gray to black. Conidia were black, spherical to subspherical, and unicellular, measuring 14.6 ± 1.7 μm in diameter (n = 50) (Fig. 3). Based on these cultural and morphological characteristics, isolate YBB-5 was preliminarily identified as Nigrospora spp., which is in agreement with the description of this species (Wang et al. 2017). To further confirm the identity of the isolate, genomic DNA was extracted from isolate YBB-5, and the internal transcribed spacer (ITS) region was amplified using the primer pair ITS1/ITS4. The obtained ITS sequence was deposited in GenBank under accession number PZ574363. For phylogenetic analysis, ITS sequences of representative Nigrospora species and related taxa were retrieved from GenBank. Multiple sequence alignment and phylogenetic analyses were conducted using MEGA version 11.0. A neighbor-joining phylogenetic tree was constructed based on the Tamura–Nei model with 1,000 bootstrap replications. Isolate YBB-5 clustered with N. sphaerica isolate QYN6 (PV336075.1) with strong bootstrap support (99%) and was clearly separated from other Nigrospora species included in the analysis. These results, in conjunction with morphological characteristics, confirmed the identification of the isolate as N. sphaerica (Fig. 4). To assess pathogenicity, mycelial plugs (5 mm in diameter) obtained from the margin of 7-day-old PDA cultures of isolate YBB-5 were placed onto 15 detached healthy yam leaves (five leaves per replicate, three replicates) after surface sterilization. Fifteen detached healthy leaves treated with sterile PDA plugs served as controls. The inoculated leaves were maintained in sterile Petri dishes containing moist filter paper and incubated at 25 ± 1°C under a 12 h light/dark photoperiod. The experiment was repeated three times. At 3–5 dpi, typical symptoms, resembling those observed in the field, developed around the inoculation sites, whereas the control leaves remained symptomless. To further verify pathogenicity under natural conditions, 30 healthy yam leaves were slightly wounded with sterile syringes and sprayed with a conidial suspension obtained from cultures grown in potato dextrose broth (PDB) at 27°C for 7 days. Thirty healthy leaves sprayed with sterile water served as controls. Approximately 14 days after inoculation, inoculated leaves developed characteristic leaf spot symptoms consisting of grayish-white necrotic centers surrounded by dark-brown margins, closely resembling those observed under natural conditions, whereas control leaves remained symptomless(Fig. 5). The fungus was consistently re-isolated from symptomatic tissues. The recovered isolates exhibited cultural and morphological characteristics matching those of the original isolate, including the production of typical black spherical conidia. These results fulfilled Koch’s postulates. No obvious symptoms were observed on tubers, and no obvious differences in tuber size or individual tuber weight were noted among the limited number of plants examined; however, the effect of the disease on overall tuber yield was not quantitatively evaluated. Nigrospora sphaerica has previously been reported to cause leaf spot or leaf blight on watermelon, passion fruit, cacao, and peach (Ismail and Abd Razak 2021; Li et al. 2024; Villanueva et al. 2023; Wang et al. 2022). These diseases are associated with extensive foliar necrosis, premature defoliation, and reduced yield. To our knowledge, this is the first report of N. sphaerica causing leaf spot on yam in China. This finding extends the known host range of N. sphaerica and provides a basis for future monitoring and disease management in yam production systems.