First Report of Nigrospora sphaerica Causing Leaf Blight Disease on Amorphophallus bulbifer in Shaanxi Province of China

Amorphophallus bulbifer (Roxb.) Blume is a perennial tuber that belongs to the family Araceae, is widely used in food, pharmaceutical, and chemical industries because of its richness in glucomannan. It is mainly distributed in southern Yunnan of China, northern Myanmar, Laos, northern Thailand and tropical rain forest areas in Indonesia (Gao et al, 2022). In July to August 2023, a leaf blight disease occurred on A. bulbifer in Ankang Academy of Agricultural Sciences manufacturing base (32°69′N, 109°02′E), Shaanxi, China. About 100 plants were investigated, and the results showed that the incidence of leaf blight disease was 36%. The symptoms were characterized by yellow-brown and irregular lesions. With time, the lesions enlarged and affected leaves finally became wilted. To investigate the disease, ten plants with typical symptoms were collected, one leaf was selected from each plant, and cut into 2 mm × 2 mm pieces. Their surfaces were disinfected with 1.5% NaClO for 60s followed by 75% ethanol for 30s, rinsed three times with sterile distilled water, and finally incubated on 1.5% potato dextrose agar (PDA) plates at 28℃. A total of six isolates were obtained through single-spore cultivation. The colonies on PDA were fluffy with abundant aerial mycelia and covered the whole plates (90 mm in diameter) in seven days. Fungal colonies were white, then turned to grayish with an unevenly distributed (Fig.1f-g). Conidia were single-celled, black, smooth, spherical to subspherical, 15-18 μm in diameter (Fig.1h-j). These morphological characteristics were similar to those of Nigrospora sphaerica (Wang et al.2017). For molecular confirmation, genomic DNA was extracted from mycelia of the pathogen using an Ezup column fungal genomic DNA extraction kit (Sangon Biotech, Shanghai, China). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS), TEF-1 and TUB2 gene of the isolate were amplified by PCR (Duan et al., 2024) and sent for sequencing. The resultant sequence (GeneBank ID of gene ITS, TEF-1, TUB2 are PQ330916, PQ778340, PQ778339, respectively) were compared with the voucher specimens. BLAST results showed >99% identity with those of Nigrospora sphaerica (GeneBank ID of Nigrospora sphaerica strain LC7298 ITS, TEF-1, TUB2 are KX985937, KY019401, KY019606, respectively). A neighbor joining phylogenetic tree with the concatenated sequences of these genes showed that Ab-B23 had the closest match with Nigrospora sphaerica (Fig. 2). Based on morphological characteristics and molecular dataset analyses, the isolates were identified as Nigrospora sphaerica. For pathogenicity test, twenty healthy 1-year-old Amorphophallus bulbifer plants were disinfected with 75% alcohol before inoculation, then sprayed with conidial suspensions (100 µL, 1×10⁶ conidia/ml sterile water) of the isolates; the other twenty plants, also disinfected with 75% alcohol before inoculation, were sprayed with sterile water as the controls. All the treated plants were cultivated in a glasshouse at 28±2℃ and 80 ± 5% relative humidity. Seven days after inoculation, a yellow-brown spot appeared on leaf surfaces, and the spot gradually expanded. Fifteen days after inoculation, infected leaves showed yellow-brown and irregular lesions, whereas the control plants remained symptomless (Fig.1a-e). The pathogen was reisolated from infected leaves with an 85% reisolation percentage, and was confirmed as Nigrospora sphaerica by morphology and molecular identification. To our knowledge, this is the first report of leaf blight disease of Amorphophallus bulbifer caused by Nigrospora sphaerica in Shaanxi Province of China. Our report would be helpful to Amorphophallus bulbifer growers to recognize this leaf blight disease, and corresponding measures could be taken to minimize or avoid the economic losses caused by the disease.