Leaf Spot Caused by Didymella segeticola on Tobacco in China

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. In China, the seed area of tobacco was over 14,000 km², and the yield of tobacco leafage increased to 31.32 million tons in 2011 (Meng et al. 2015). In August 2019, diseased leaves of tobacco that had sandy beige with dark-brown edge, circular, elliptical or irregular shaped lesions and surrounded by yellow halos were obtained (cv. Yunyan 87) in Shibing (27.03° N, 108.11° E), Guizhou, China. Diseased leaf segments were surface sterilized and plated on potato dextrose agar (PDA). Isolate YBZ121 was selected for identification. The colonies had dense aerial hyphae, initially white and later producing gray near the center when cultured on PDA. The colonies had woolly aerial hyphae, white to gray eventually, and produced pycnidia on oatmeal agar (Boerema et al. 2004). Pycnidia were dark, spherical or flat spherical, and 80.3 to 148.4 µm in diameter. Conidia were oval mostly, smooth, aseptate, usually guttulate, and the size was 3.8 to 4.9 × 1.9 to 2.7 µm. The rDNA internal transcribed spacer region (ITS) with primers ITS1f/ITS4 (Gardes and Bruns 1993; White et al. 1990), 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 YBZ121 were sequenced (GenBank accession nos. MN639735, MN639771, MN640785, and MN640786, respectively). A phylogenetic tree based on these four concatenated sequences showed that YBZ121 comprised a clade with Didymella segeticola (syn. Phoma segeticola) strain CGMCC 3.17489 with 98% bootstrap support. Tobacco plants at seedling stage (six to seven leaves) without visible disease were used for the pathogenicity test. Sterile filter papers (5 mm in diameter) were immersed in conidial suspension (10⁶ spores/ml) containing 0.01% Tween 80 for 1 min and then placed on leaves of tobacco plants, with sterile water containing 0.01% Tween 80 used as a noninoculated control. One leaf of tobacco plant was inoculated with four pieces of filter paper containing spores and one inoculated with no spores, with three replicates per treatment, and then put in a plant incubator at 28°C with a 12/12 h light/dark cycle. Nine days after incubation, typical symptoms were observed on inoculated leaves but not on control leaves. Koch’s postulates were fulfilled by reisolation of D. segeticola from diseased leaves. Based on morphological and multigene molecular data, isolate YBZ121 was identified as D. segeticola Q. Chen, sp. nov. described as a new taxon by Chen et al. (2015). It has been reported to cause leaf spot on tea in China (Zhao et al. 2018). However, to our best knowledge, this is the first report of D. segeticola causing leaf spot on tobacco in China. D. segeticola has the potential to cause considerable economic loss to farmers. Disease management of leaf spot caused by D. segeticola on tobacco needs to be developed to reduce losses in the future.