First Report of <i>Nigrospora musae</i> Causing <i>Idesia polycarpa</i> Leaf Spot in China

Idesia polycarpa Maxim. was one of unique tree species in East Asia and was chiefly cultivated between subtropical and warm temperate zones, with potential medicinal properties (Kim et al. 2005). In June 2023, leaf spot symptoms on I. polycarpa were observed in Guiding County (26°33'N, 107°13'E), Guizhou Province, with a incidence rate of 62% (n=150). Initial symptoms manifested as circular to irregular brown necrotic spots featuring pronounced dark brown margins and diffuse chlorotic halos. As the disease progressed, lesions expanded to form extensive necrotic patches. In some severe cases, the whole leaf turned to necrosis, ultimately causing premature defoliation. Symptomatic leaves (n=3) were collected and surface disinfected with 75% ethanol for 30 s, 1% NaClO for 30 s, rinsed 3 times in sterile water, air-dried and placed on potato dextrose agar (PDA) medium and incubated at 26°C for 5 d. Fungal colonies on PDA of three similar obtained isolates were white and massive, with sparse aerial hyphae that became dark brown when incubated for 10 to 14 d at 26 °C. Microscopic observation shows that the hyphae are transparent, branched, and brown in color. Conidia were solitary, smooth walled, black, and globose to sub-globose with a diameter of 17.75±1.29 μm (n=30). Based on morphological, cultural and microscopic characteristics, the isolates were found similar to Nigrospora musae reported by Liao et al (2023). For molecular identification, DNA of the representative isolates ST1, ST2, ST3 was extracted using a DNA extraction kit (Sangon, Shanghai, China). The rDNA internal transcribed spacer (ITS), translation elongation factor 1-α (TEF-1α) and β-tubulin (TUB2) were amplified with the primers ITS1/ITS4 (White et al.1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999) and Bt2a/Bt2b (Glass and Donaldson, 1995). The sequences of ITS, TEF-1α and TUB2 were deposited in GenBank (ST1: PQ549970, PQ559741 and PQ559744; ST2: PQ549971, PQ559742 and PQ559745; ST3: PQ549972, PQ559743 and PQ559746). BLAST results showed the sequences of ST1 were highly identical to those sequences of N. musae (NR153478 [ITS], 519/520; OP450784.1 [TEF-1α], 290/292; OP450804.1 [TUB2], 430/432). The phylogenetic tree constructed with the software MEGA X using Maximum likelihood method indicated the isolate ST1, ST2 and ST3 gathered into one branch with N. musae CBS 319.34. Based on these results, the three isolates were identified as N. musae. To confirm pathogenicity, spore suspensions (1×106 spores/mL) of ST1 were sprayed on healthy detached leaves (n=3) and leaves of 2-year-old potted I. polycarpa seedlings (n=3). An equal volume of sterile water was sprayed for the control. Then, they were cultured in a greenhouse at 26 ± 2 °C and 80% relative humidity. The inoculated leaves and seedlings showed similar leaf spot symptoms within 4~7 d. The re-isolated fungus, was identical to N. musae based on morphological and molecular analysis, thus fulfilling Koch's postulates. N. musae has been already reported as a plant pathogen from China from Basella alba (Liao et al. 2023) and Taxus chinensis var. mairei (Chen et al. 2023). But this is the first report of N. musae causing leaf spot of I. polycarpa in China, supporting a theoretical basis for effective prevention and control of this disease.