Leaf Spot on Alocasia macrorrhizos Caused by Fusarium asiaticum in Sichuan, China

Alocasia macrorrhizos (giant elephant’s ear), a perennial herb in the Araceae family, is native to South Asia and the Asia-Pacific (Takano et al. 2012). It is cultivated as a medicinal and ornamental plant and has considerable economic importance in China. In September 2020, a severe infection of an unknown leaf spot disease was observed on these plants at Sichuan Agricultural University, Sichuan, China. The leaf spots first appeared as yellow dots. As the lesions expanded, they became circular to oval and light brown with darker brown edges. Around the lesions, the leaf tissue was chlorotic, creating a yellow halo. When the infection became severe, spots merged into larger irregular lesions. Eventually, the diseased leaves senesced and dried. To identify the pathogen, five leaf samples of diseased plants were collected, and symptomatic tissues were surface disinfected with 75% ethanol for 30 s followed by 3% NaCl solution for 30 s. Samples were rinsed three times in sterilized water, placed on potato dextrose agar (PDA), and incubated at 25 ± 1°C in the dark. The colony grown on PDA was white (3 days), the center was brown (5 days), and turned pink to dark red (8 days) with fluffy aerial mycelium and pigmentation with age. Ten pure cultures were inoculated into carnation leaf agar (CLA) medium and incubated at 25°C in an incubator (12 h for one light-dark cycle). In CLA medium, the pathogen produced hyaline, sickle-shaped macroconidia with three to five septa and an average size of 30 to 50 × 4 to 5 μm (n = 30), but no microconidia in 10 days. Chlamydospores were spherical to subspherical (5.4 to 13.8 μm). Morphological characteristics of the all isolates were consistent with the description of Fusarium asiaticum (Leslie and Summerall 2006). To validate this identification, RNA polymerase II (RPB2) (Liu et al. 1999), translation elongation factor (EF-1) (Geiser et al. 2004), and β-tubulin (TUB2) gene regions of five isolates were amplified and sequenced (O’Donnell and Cigelnik 1997; White et al. 1990). The sequence of one representative isolate (ZL10) was submitted to GenBank (ON215729, ON215730, and ON215731). NCBI BLAST identified the top hits, with 100, 100, and 99.87% identity for RPB2, EF, and TUB gene sequences, respectively, all indicating F. asiaticum. Pairwise matching of RPB2 and EF genes by MycoBank Fusarium MSIL showed the top hit rate of 100% for F. asiaticum (MH582120 and MH582249). For Koch’s postulates and a pathogenicity test, leaves on 1-year-old A. macrorrhizos plants were inoculated with spore suspensions (1 × 10⁷ conidia/ml) collected from PDA and CLA cultures with 0.05% Tween 80 buffer using a spray bottle. Two leaves of each plant (20 pots in total) were inoculated with the spore suspension (approximately 2000 μl per leaf). An equal number of control leaves were sprayed with water and 0.05% Tween 80 buffer. Twenty days later, the inoculated plants showed similar symptoms to those of the original diseased plants while the controls remained asymptomatic. F. asiaticum was reisolated from the infected leaves and confirmed using morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. This first report raises awareness of a new leaf spot disease infecting commercial A. macrorrhizos in China. It shows the need for a systematic survey identifying the current spread, disease origin, and ultimately developing disease management strategies.