Tobacco (Nicotiana tabacum L.), is a major cash crop grown worldwide for its leaves, which are dried and fermented before being put in tobacco products. Tobacco production is seriously threatened by numerous diseases (Qiu et al. 2021). In August 2021, plants with stem-end rot were observed in a tobacco field in Zunyi City, Guizhou Province, China. Surveys indicated a 22 to 35% disease incidence in five counties. Symptoms of black necrosis appeared at the base of stems, and leaves turned yellow. To isolate the pathogen, diseased stems were cut into small segments and placed on potato dextrose agar (PDA) at 25°C in darkness for 3 to 5 days. To obtain pure cultures, hyphal tips from colonies were transferred to fresh PDA plates. A representative strain, GZAX 110, was used for further identification. The fungal colonies were initially gray, later deepening to smoke-gray. Conidiogenous cells were fully divided, discrete, transparent, thin-walled, smooth and cylindrical. Conidia matured slowly, were ellipsoid to ovoid, containing granular content, with a rounded apex. The base was largely truncated, and conidia became dark brown with one central septum, 21.0-30.0 × 12.0-18.0 μm. On water-agar medium, teleomorph structures were not observed. These characteristics suggested the fungus was Lasiodiplodia sp. (Netto et al. 2014). For further confirmation, genomic DNA was extracted using the CTAB method (Watanabe et al. 2010); and the ITS (internal transcribed spacer region of rDNA) and EF-1α (translation-elongation factor) regions were amplified with primer pairs ITS1/ITS4 and EF1-688F/EF1-1251R (Cruywagen et al. 2017), respectively. The ITS and EF-1α sequences (OM534558 and OM632673) were analyzed by BLASTN searches. The ITS sequence showed 100% identity (490/490 bp) to L. brasiliense strain AGQMy0011 (MW274145) and the EF-1ɑ sequence showed 100% identity (551/551 bp) to L. brasiliense strain EX1 (MF580811). A multilocus phylogenetic tree was constructed via the Maximum-likelihood (RAxML v.7.2.8) and Bayesian Inference (MrBayes v.3.2.1) analyses (Elsie et al. 2017) using combined ITS and EF1-α reference sequences of Lasiodiplodia species. Phylogenetic analysis showed that GZAX 110 clustered monophyletically with strains of L. brasiliense. Thus, the isolate GZAX 110 was confirmed as L. brasiliense. Pathogenicity of GZAX 110 was tested on tobacco plants at the eight leaf stage by attaching mycelial plugs (5 mm in diameter) to stems and leaves according to Cruywagen et al. (2017). Inoculated plants were kept in a greenhouse (16 h light/8 h darkness, 22℃, relative humidity >85%). Control plants were inoculated with PDA plugs. The experiment was repeated three times with five plants. Seven days after inoculation, dark brown necrosis was observed at inoculation sites on stems and leaves, while the control plants remained healthy. The pathogen was re-isolated from the inoculated sites and further validated as the same fungus through morphological and phylogenetic analyses. Previously, this fungus has been reported on Mangifera indica (mango) in China (Zhang et al. 2018), and apple (Martins et al. 2018) and papaya (Netto et al. 2014) in Brazil. However, to our knowledge, this is the first worldwide report of L. brasiliense causing stem-end rot on tobacco. This report provides information for future diagnosis and management of the disease.
Keywords: Lasiodiplodia brasiliense; Stem-end Rot; Tobacco.