First report of Fusarium odoratissimum causing root rot of tobacco in Huanghuai region of China

In recent years, Fusarium root rot has emerged as a significant root disease, leading to substantial yield losses in Huanghuai tobacco-growing region of China. In June 2025, a survey was conducted across approximately 267 hectares of tobacco (cv. Zhongyan100) fields in seven cities (Weifang, Linyi, Xuchang, Zhumadian, Luoyang, Sanmenxia, and Xinyang) within the Huanghuai region. Tobacco root rot incidence ranged from 20% to 50%. Affected plants exhibited wilting and basipetal leaf chlorosis. Uprooted specimens displayed severe root rot, black necrotic lesions on roots, and brown to purplish vascular discoloration. To isolate the pathogen, small tissue segments (approximately 5 mm) were excised from the margins of lesions on the roots of 40 randomly collected symptomatic plants. The tissues were surface-sterilized in 75% ethanol for 30 s, followed by immerse in 2% NaClO for 10 min, rinsed three times with sterile distilled water, and plated on half-strength potato dextrose agar (½PDA). The plates were incubated at 25℃ in the dark for 3 days. Thirty-five pure cultures were obtained by single-spore isolation, among which the dominant colonies (65.71%) were initially white and later developed a pinkish center, with cottony aerial mycelium, and an average growth of 40.67 ± 0.76 mm in three days on PDA. On synthetic nutrient-poor agar (SNA) or carnation leaf agar (CLA), macroconidia were falcate to falciform, with slightly curved hoot-like apical cell and a distinct foot cell at the base, 2- to 3-septate, and measured 23.93 × 3.08 to 37.80 × 4.08 μm (mean=27.58 ± 2.16 μm × 3.35 ± 0.16 μm, n=50). Abundant reniform, elliptical, or cylindrical microconidia were one to two-celled, with apexes rounded, measuring 3.51 to 23.10 μm × 1.53 to 4.15 μm (n=50). Spherical conidia clusters were formed at the apex of the conidiophores. Chlamydospores were roughly spherical, single, terminal, and rough-walled, 8.36 ± 0.26 μm (Fig.1). For molecular identification, genomic DNA was extracted from mycelia of the 23 dominant isolates. Fragments of the translation elongation factor 1-alpha (tef1), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) genes were amplified with primer pairs EF1/EF2, RPB2F/R, and F5/G2R, respectively (O’Donnell et al. 1998, 2010). BLASTn analysis in FUSARIOID-ID database showed that the sequences of the tef1 (GenBank accession no. PZ142847-PZ142882), RPB1 (GenBank accession no. PZ142953-PZ142988), and RPB2 (GenBank accession no. PZ143059-PZ143094) regions shared 99.88% to 100% identity with those of Fusarium odoratissimum reference strains (e.g., NRRL 54006). A phylogenetic tree constructed based on the combined tef1, RPB1, and RPB2 sequences placed the isolates in a clade with F. odoratissimum with 96% bootstrap support (Fig.2). Morphological and molecular results confirmed these species as F. odoratissimum (Leslie and Summerell 2006; Tamang et al. 2024). To verify pathogenicity according to Koch’s postulates, healthy five-leaf-stage tobacco seedlings (cv. Zhongyan 100) were used. Spore suspensions (5 × 106 conidia/mL) from 7-day-old cultures were inoculated into the stem bases of 30 seedlings. Another 30 seedlings treated with sterile distilled water served as the control group. All treatments were maintained in a growth chamber at 28℃ with 70% relative humidity. After 21 days, inoculated plants developed symptoms identical to those observed in the field, including foliage chlorosis and root rot, whereas control plants remained healthy. The fungus were consistently reisolated from the symptomatic tissues of inoculated plants and confirmed morphologically and molecularly as F. odoratissimum, whereas no pathogen was recovered from control plants. Fusarium odoratissimum is recognized as a major causal agent of banana Fusarium wilt (Tamang et al. 2024). It is also capable of infecting a wide range of hosts, including cereal crops, fruits, and forest trees (Ma et al. 2025; Wang et al. 2026; Liu et al. 2025). To our knowledge, this is the first report of F. odoratissimum causing root rot on tobacco in Huanghuai region of China. The accurate identification of this pathogen will provide a theoretical basis for the development of effective green control strategies against this disease.