First Report of Stem Base Rot Disease on Tobacco Caused by <i>Didymella heterodera</i> in China

In February 2025, a disease affecting tobacco seedlings was identified in Chenzhou, Hunan Province, China. Affected plants exhibited stunted growth, with yellowing leaves at the lower portions and blackish-brown spots appearing at the stem base. Subsequently, stem decay caused plant collapse, leading to the death of the entire seedling. Approximately 20% of tobacco seedlings were affected across ten cultivation greenhouses, which collectively housed nearly 3.5 million seedlings. Symptomatic stem tissues were sterilized in 70% ethanol for 10 s, followed by treatment with 3% NaClO for 2 minutes. The tissues were then washed three times with sterile distilled water and placed on potato dextrose agar (PDA) plates for incubation at 26°C. Eight isolates with similar morphology were selected from eight plants and sub-cultured on PDA. The colonies initially appeared whitish and gradually turned gray, developing dense aerial hyphae. After more than ten days of incubation, black, spherical pycnidia measuring 18.2-37.8 µm × 18.2-30.7 µm (n = 50) were produced. The conidia were oval to oviform, smooth, and aseptate, measuring 3.6 to 6.9 µm × 2.3 to 3.8 µm (n = 50). The morphological characteristics were consistent with those of the Didymellaceae species (Chen et al. 2017). For molecular identification, the partial 28S large subunit rDNA (LSU), internal transcribed spacer (ITS), second-largest subunit of RNA polymerase II (RPB2), and β-tubulin (TUB2) genes of two representative isolates were amplified and sequenced using primer pairs LROR/LR7 (Rehner and Samuels, 1994; Vilgalys and Hester, 1990), ITS4/ITS5 (White et al. 1990), RPB2-5F2/RPB2-7-cR (Sung et al. 2007; Liu et al. 1999), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), respectively. As the sequences were identical, we selected isolate CZ-YG-1 for further analysis, with accession numbers PV697019, PV697020, PV705038, and PV705039 for LSU, ITS, RPB2, and TUB2, respectively. BLASTn searches against the NCBI database showed 99 to 100% identity with corresponding sequences of Didymella heterodera strains (1324/1324 bp, GU238002.1 for LSU; 494/494 bp, LT592922.1 for ITS; 937/937 bp, LT593056.1 for RPB2; 362/364 bp, LT592987.1 for TUB2). A phylogenetic tree from concatenated LSU, ITS, RPB2, and TUB2 sequences revealed that isolate CZ-YG-1 clustered with D. heterodera strain CBS 109.92, with 100% bootstrap support. For the pathogenicity test, a conidial suspension was prepared by harvesting conidia from 14-day-old PDA cultures and adjusting the concentration to 1 × 107 conidia/mL. Potted tobacco seedlings of the cultivar Yunyan 87 were inoculated with CZ-YG-1 conidia by root irrigation, with each plant receiving a volume of 5 mL. Plants treated with sterile water served as controls. The pathogenicity assay was repeated three times, with five replicates per treatment. All inoculated plants were placed in humid chambers maintained at 90% humidity and 26°C, with a 12-hour light/12-hour dark cycle. Seven days after inoculation, the inoculated plants exhibited typical symptoms of collapse and brown necrosis at the stem bases, whereas the control plants showed no symptoms. The pathogenic fungus was re-isolated and identified using morphological and molecular methods, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of D. heterodera causing tobacco stem base rot in China. This report may provide valuable insights for the diagnosis and regional management of this disease on tobacco.