First report of tobacco root rot caused by Fusarium oxysporum and Fusarium solani infection in Northeast China.

Tobacco root rot caused by Fusarium spp. infection results in significant losses to tobacco production. The disease is primarily prevalent in southern and central China, particularly in Fujian, Shandong, and Shanxi (Qiu et al. 2018). In July 2023, tobacco samples (n=10) exhibiting root rot symptoms were collected from Maodianzi Town (40.67°N, 124.62°E; cv. Liaoyan 21) and Qingyishan Town (40.68°N, 124.62°E; cv. Liaoyan 21) in Kuandian, Liaoning Province, China. The disease incidence in the surveyed fields reached approximately 60% to 70% based on the five-point sampling method. The symptoms exhibited blackening and rotting of the main root, stem base, and sparse fibrous roots. To isolate and identify the pathogen, the diseased tissues (2 × 2 mm) were cut from the edge of root lesions, treated with 75% alcohol rinsed with sterile water, and then incubated on potato dextrose agar (PDA) medium at 28 ℃. A total of 21 isolations were obtained through the single-spore isolation method. The experiment was conducted in a laminar flow cabinet and replicated three times. Two morphological types were observed among the colonies and spores of these strains. One type exhibited white, fluffy colonies. Large conidia were sickle-shaped, slightly rounded at both ends, and mostly had two to five septa (16.2×2.1 μm). Small conidia were spindle-shaped to ovate (8.2×2.3 μm) and generally had one septum (Qiu et al. 2024). Another type showed colonies with pink and slightly purple hues, and the spores were sickle-shaped, slightly curved spores with pointed somewhat ends, small conidia were mostly without septa or one septum (15.1×2.0 μm), and large conidia were unequally septate or sickle-shaped (39.4×4.2 μm), mostly with three to five septa (Sang et al. 1998). The mycelia of both fungal isolates had a small number of branches. Two isolates (GF11, GF12) were selected from two different morphologies, and subjected to DNA extraction using the CTAB method (Sagar et al. 2014). Sequencing analysis of the two strains was performed using the nuclear ribosomal internal transcribed spacer region (ITS, GenBank accession no. PQ608542 and PQ608543), translation elongation factor (TEF-1a, PQ671287 and PQ671288), and the second largest subunit of RNA polymerase II (RPB2, PQ671289 and PQ671290) (O'Donnell et al. 2000, 2015). Upon comparison with NCBI database sequences, the ITS and EF-1α sequences of strain GF11 were found to exhibit a maximum homology of 100% with the corresponding sequences of F. oxysporum, while the RPB2 sequence showed a maximum homology of 99.66%. For strain GF12, its ITS, EF-1α, and RPB2 sequences were determined to have maximum homologies of 100%, 99.72%, and 99.55% respectively with the corresponding sequences of F. solani. According to Koch's postulates, GF11 and GF12 were used for pathogenicity testing. Nicotiana tabacum cv. K326 were cultivated at the 26°C growth chamber and used at the four-leaf stage. The base of the stem of ten plants was wrapped in sterile cotton treated with a spore suspension (1×10⁶ conidia/mL) of GF11 and GF12. The control group was treated with sterile water. After 10 days, the GF11 and GF12 treated groups showed symptoms consistent with those observed in the field, with the fibrous roots becoming sparse and the leaves turning yellow, and the F. oxysporum and F. solani were successfully re-isolated and identified by sequencing. This is the first report of tobacco root rot caused by Fusarium spp. in northeast China.