First Report of Leaf spot Caused by Xylaria arbuscula on Flue-cured Tobacco in China.

Abstract

Flue-cured tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves in China. Around 70% of tobacco production in China occurs in southwest China. In summer of 2019, leaf spot symptoms were observed on ten to twenty percent of tobacco plants in a 2 ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. The leaf spots were white with dark-brown in edges, irregularly round and oval, and diseased tissue dropped out leaving the leaves ragged in appearance (Fig. 1A, 1B). One diseased leaf from each of five plants was sampled. From five leaves, a total of 15 small (5 mm × 5 mm) pieces of leaf tissue were cut from the edge of the lesions after surface sterilization and placed on potato dextrose agar (PDA) medium. Five fungal colonies that were similar in appearance were isolated and one was purified, BEZ22, was selected arbitrarily for identification. Mycelia of the pathogen was initally white and dense, and then black carbonized mycelia appeared from the center of the colony 7 days' after incubation. Mycelia was white, sparse and radiated when incubated on OA (oatmeal agar) (Fig. 1E, 1F, 1G, 1H). Genomic DNA of the isolate was extracted. The internal transcribed spacers (ITS) with primers ITS1/ITS4 (White et al. 1990), actin (ACT) gene with primers ACT-512F/ACT-738R (Hsieh et al. 2005), beta-tubulin (TUB2) with primers T1/T22 (O'Donnell & Cigelnik 1997) and RNA polymerase II second largest subunit gene (RPB2) with primers fRPB2-5F/ fRPB2-7cR (Liu et al. 1999) were amplified and sequenced, respectively. The generated sequences were deposited in GenBank with accession numbers MT804353 (ITS), MT809582 (ACT), MT799790 (TUB2) and MT799789 (RPB2). Using BLASTN searches, the sequences of each gene above were aligned with the voucher specimum, Xylaria arbuscula 89041211. The number of nucleotides that were similar for ITS (GU300090) was 550/551 (99%); for ACT (GQ421286), 266/266 bp (100%); for TUB2 (GQ478226), 1501/1501 bp (100%); and for RPB2 (GQ844805), 1135/1135 bp (100%), respectively (Fig. 2). A phylogenetic tree was constructed based on these four sequences with a final alignment of 3456 characters (ITS 551, ACT 266, TUB2 1501 and RPB2 1138). Thus, based on morphological and phylogenetic analyses, the isolate BEZ22 was identified as Xylaria arbuscula. To verify pathogenicity, six tobacco plants at seedling stage (5-6 leaves) without visible disease were inoculated using mycelial plugs (5 mm in diameter). Leaves inoculated with PDA only plugs served as controls. After inoculation, all tobacco plants were maintained in a greenhouse with 85% relative humidity at 25 oC under a 12/12 h light/dark cycle. Five days after inoculation, typical early symptoms were observed on the inoculated leaves, and not on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from diseased leaves. Xylaria arbuscula has also been reported as a pathogen of Macadamia in Hawaii (Wenhsiung et al. 2009) and sugarcane in Indonesia (Maryono et al. 2020). However, to our best knowledge, this is the first report of X. arbuscula causing leaf spot on tobacco in China. This leaf spot has the potential to cause serious damage to tobacco in this region that could result in reduced production, consequently disease management of this pathogen should be considered.