First Report of Alternaria alternata Causing Leaf Spot on Nelumbo nucifera Plants in China

White lotus (Nelumbo nucifera) is an aquatic plant of the Nymphaeaceae family that primarily serves as an ornamental plant and is an important cash crop in China. In May 2020, an unknown leaf disease affecting these plants was first detected in White Lotus Science and Technology Expo Park in Guangchang County, Fuzhou City, Jiangxi Province (26.79° N, 116.31° E). The disease caused approximately 30-40% of leaves to die, and led to 15 to 20% in seed yield losses. This disease was characterized by the formation of irregular yellow-brown to dark-brown spots during the initial phases of infection. As the disease is developing, these spots expanded until they were generally round and brown to purple-brown in color, with a yellow halo surrounding the expanding spots. In an effort to characterize the causative pathogen, a small ~5×5 mm leaf tissue section from the boundary between normal and diseased tissue was collected, and sterilized with the following regimes: 30 s with 75% ethanol, soaked in 0.1% mercuric chloride for 30 s, washed thrice with sterile water, and transferred onto potato dextrose agar (PDA) plate, and placed in an illumination incubator (12 h light/dark) at 28 °C± 1°C for 5 days. Seven pure cultures were obtained from ten diseased leaves. For pathogenicity testing, a hyphal inoculation strategy was employed, with all studies being conducted at the Plant Pathology Laboratory of Jiangxi Agricultural University. Five mm discs were selected from three separate cultures and one control (PDA). Healthy leaves of lotus seeds were treated with 4 treatments per leaf including three separate cultures and one control that were treated with the test pathogen. The experiments were repeated three times with three biological replicates. Healthy leaves were covered with moisturized sterile cotton balls and fixed to the leaf surface with transparent tape. The inoculated lotus seedlings were kept in greenhouse incubator at 28 °C± 3°C and relative humidity of 70 to 80%. Following a 14-day incubation period, brown spots began to manifest at all sites inoculated with the test pathogen whereas the control spots remained healthy. Diseased spots were then separated. The same pathogen was once again successfully isolated and identified microscopically, thus fulfilling Koch's postulates. Six isolates were characterized. Ovoid or elliptical conidia were brown to light-brown in color with a short beak, 1-5 diaphragms, and 0-3 mediastinum. The diameter of these conidia were thick (13.8-44.0×7.5-16.3 µm; average: 24.0×11.9 µm, n=50). These morphological characteristics were consistent with Alternaria alternata. The ITS4/ITS5, EF1-728F/EF1-986R, AltF/AltR, OPA10-2R/OPA10-2L, EPGF/EPGR and KOG1058F2/KOG1058R2 primer sets were then used to conduct molecular identification by amplifying key transcription elongation factor and internal transcriptional spacer regions, yielding sequences that were 99%-100% similar to Alternaria alternata (GenBank No: MK396606, MT178329, MN184998, MN894688, MT849815 and KP125234). Sequences were deposited in GenBank with accession numbers MW898580 (ITS, 620 bp), MW981281 (EF1-α, 284 bp), MZ514094 (Alt a1, 477 bp), MZ514095 (OPA10-2, 716 bp), MZ514096 (endoPG, 465 bp) and MZ514097 (KOG1058, 877 bp). Nelumbo nucifera is an important aquatic cash crop in China, and this is the first study we are aware of demonstrating the presence of a leaf disease caused by Alternaria alternata in Nelumbo nucifera plants anywhere in the world. These findings may offer a foundation for efforts to prevent diseases caused by Alternaria alternata.