Lophatherum gracile Brongn. is an important Chinese herbal medicine. Since 2016, a leaf spot disease has appeared on L. gracile seedlings in the traditional Chinese medicine resource garden of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province (32.06°N, 118.83°E). About approximately 80% of the seedlings suffered from the disease. The disease spot usually starts from the leaf margin, round or irregular, with yellow halo at the edge of the lesion. To isolate the pathogen, four diseased leaves were collected from four different seedlings and there are 6 sections from each diseased leaf. The leaf sections were surface sterilized in 75% alcohol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA). Pure cultures were obtained by monosporic isolation. Eleven isolates were obtained (isolate rate of 55%) and identified as Epicoccum sp.. Thus, a representative isolate, DZY3-3 was used for the further study. After 7 days of culture, the colony produced white aerial hyphae, and reddish orange pigment on the underside. The chlamydospores were produced, either multicellular or unicellular. The colony produced pycnidia and conidia after nearly three weeks of cultivation on oatmeal ager OA. Conidia were unicellular, hyaline, oval, and were 4.9 to 6.4 x 2.0 to 3.3 μm (n=35). In addition, a brown discoloration was produced on malt extract agar (MEA) after using the 1 mol/L NaOH solution for 1 h. These characteristics were consistent with the description of Epicoccum sp. (Chen et al. 2017). To comfirm this identification, the internal transcribed spacer (ITS), large subunit ribosomal RNA (LSU), beta-tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) regions were amplified using the detailed primer pairs described by White et al., Rehner and Samuels, Woudenberg et al. and Liu et al., respectively. They had 99.8-100% homology to the ITS (GenBank no. MN215613, 504/505 bp), LSU (MN533800, 809/809 bp), TUB (MN329871, 333/333 bp), and RPB2 (MG787263, 596/596 bp) sequences of E. latusicollum in the GenBank database. A neighbor-joining phylogenetic tree was generated based on the concatenated sequences of all the above regions in MEGA7. The DZY3-3 clustered in the E. latusicollum clade with 100% bootstrap support. Koch's postulates were performed by spray inoculation (1×106 spores/mL) on the left sides of leaves of three healthy L. gracile seedlings and detached leaves, using isolate DZY3-3, while sterilized water served as the control was sprayed on the right sides of leaves. All plants and detached leaves were covered with clear polyethylene bags to maintain about 80% relative humidity at 25℃. Whether in vivo or in vitro pathogenicity test showed similar symptoms to those occurred in the field after 5 days post inoculation. No symptoms occurred on controls. The experiment was repeated three times. Subsequently, the same fungus was reisolated and identified from leaves of three inoculated seedlings. The E. latusicollum has a very wide host range. For example, it has been reported to cause stalk rot on Maize (Xu et al. 2022) and cause leaf spot on Tobacco in China (Guo et al. 2020). To our knowledge, it is the first report of E. latusicollum causing leaf spot on L. gracile in the world. This study will provide an important reference for the biology of E. latusicollum and the distribution of the disease.
Keywords: Epicoccum latusicollum; Lophatherum gracile; leaf spot; pathogen identification.