Zheng Z, Li Y, Wang M, Ruan R, Yang B, Zhu T, Li S, Liu Y, Qiao T, Lin T, Yang C, Li S, Huang Q, and Han S
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 a considerable economic importance in China. In September 2020, a severe infection of unknown leaf spot disease was observed on these plants at the Sichuan Agricultural University, Sichuan, China. The leaf spots first appeared as yellow dots. As these lesions expanded, they became circular to oval and light brown with darker brown edges. Around the lesions, the leaf tissue was chlorotic, thereby 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°C ± 1°C in the dark. The colony grown on PDA was white (3 days), the center was brown (5 days), 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, pathogen produced hyaline, sickle-shaped, macroconidia with 3 to 5 septa, and an average size of 30 to 50 × 4 to 5 µm (n = 30) macroconidia 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 the Fusarium asiaticum (Leslie and Summerell 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 region of five isolates were amplified and sequenced (O' Donnell et al. 2015; White et al. 1990). The sequence of one representative isolate (ZL10) sequence was submitted to GenBank (ON215729, ON215730, and ON215731). The NCBI BLAST identified the top hits, 100%, 100%, and 99.87% for RPB2, EF, and TUB gene sequences, respectively, all indicating to Fusarium asiaticum. Pairwise matched 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 postulate and pathogenicity test, spore suspensions (1 × 10^7 conidia/ml) collected from PDA and CLA cultures with 0.05% Tween 80 buffer were used to inoculate with a spray bottle on leaves of a one year old A. macrorrhizos plants. 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 applied 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. Fusarium 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 a commercial A. macrorrhizos in China. It provides an insight for a need of systematic survey identifying current spread, disease origin, and ultimately developing disease management strategies. Funding: Funding was provided by Sichuan Agricultural University Subject Dual Support Program (Grant No. 2121993055). Funding was provided by Deyang Science and Technology Bureau (Sichuan Province) for key R&D projects in agriculture and rural areas (Grant No. 2021NZ048). Funding was provided by the Sichuan Provincial Department of science and technology for the Sichuan Provincial Science and technology project for connecting and Promoting Rural Revitalization (Grant No, 2022ZHXC0007) References： Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110:473. https://doi.org/10.1023/B:EJPP.0000032386.75915.a0 Crossref, ISI, Google Scholar Leslie, J. F., and Summerall, B. A., eds. 2006. Page 176 in The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. https://doi.org/10.1002/9780470278376 Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16:1799. https://doi.org/10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., and Cigelnik, E. 1997. Mol. Phylogenet. Evol. 7:103. https://doi.org/10.1006/mpev.1996.0376 Takano K T, et al. 2012, Plant Bio., 14(4). https://doi.org/10.1111/j.1438-8677.2011.00541.x.