Leaf Spot Caused by Epicoccum latusicollum on Tobacco in China

Tobacco ( Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves in China. In continuing research on foliar diseases of tobacco in Guizhou province in August 2019, diseased leaves of tobacco that had sandy beige, elliptical or irregular shaped lesions, with brown in edge, and surrounded by yellow halos on 40% of leaves on 5% plants were obtained (cv. Yunyan 87) in Zhenan (28.55° N, 107.43° E), Guizhou, China (Fig. 1A, 1B). Diseased leaf segments were surface sterilized and plated on potato dextrose agar (PDA). Isolate (T41) was selected for identification. The colonies had white aerial hyphae, with orange-red on the underside when cultured on PDA (Fig. 1G, 1H). The colonies had woolly aerial hyphae, white to grey eventually, and produced pycnidia on oatmeal agar (OA) (Boerema et al. 2004) (Fig. 1I, 1J). Pycnidia were dark, spherical or flat spherical, and 69.2-178.0 µm in diameter. Conidia were oval mostly, aseptate, usually guttulate, and the size was 5.0 - 6.5 µm × 3.2 - 5.4 µm (Fig. 1K, 1L). The rDNA internal transcribed spacer region (ITS) with primers ITS1f/ITS4 (White et al. 1990; Gardes and Bruns 1993), 28S ribosomal RNA gene ( LSU ) with primers LROR/LR7 (Rehner and Samuels 1994), beta-tubulin gene ( TUB2 ) with primers Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RNA polymerase II second largest subunit gene ( RPB2 ) with primers RPB2-5F2/fRPB2-7cR (Liu et al. 1999) of T41 were sequenced (GenBank accession numbers were MN704804, MN710367, MN718012 and MN718013, respectively). Maximum Likelihood (ML) analyses and Bayesian Inferences (BI) analyses based on concatenated these four sequences were conducted with RAxML v. 7.2.6 and MrBayes v. 3.2.1, respectively, which showed that T41 comprised a clade with Epicoccum latusicollum strains (CGMCC 3.18346 and LC 8153) (ML/BI = 100/1) (Fig. 2). Based on morphological and multi-gene molecular data, isolate T41 was identified as E. latusicollum described as a new taxon by Chen et al. (2017). To verify pathogenicity, tobacco plants at seedling stage (7-8 leaves) without visible disease were inoculated using conidial suspension (10 6 spores/ml), following Guo et al. (2020). All inoculated plants were maintained in a greenhouse with relative humidity ranging from 50% to 85% at 28 °C under a 12/12 h light/dark cycle. Seven days after incubation, typical symptoms were observed on inoculated leaves but not on control leaves (Fig. 1C, 1D, 1E, 1F). Koch's postulates were fulfilled by re-isolation of E. latusicollum from diseased leaves. E. latusicollum has been reported to cause black root on yam in China (Han et al. 2019). Meanwhile, there are many plants could be caused leaf spot by this genus, such as Lablab purpureus (Mahadevakumar et al. 2014) and Bletilla striata (Zhou et al. 2018). However, to the best of our knowledge, this is the first report of E. latusicollum causing leaf spot on tobacco in China. Because considerable loss occurred due to infection from E. latusicollum on tobacco leaves, this pathogen is worthy of further study and disease management practices need to be developed to prevent further losses.