First report of Fusarium tricinctum Species complex causing root rot of tobacco in China

Tobacco ( L.) is an economically important crop in China. In June 2021, a root rot disease was observed on tobacco (cv. Yunyan99) in Lushi, Mianchi, and Luoning counties of western Henan, China. Diseased tobacco plants exhibited wilting with leaf chlorosis and root rot accompanied by purplish to brown vascular discoloration. The symptoms were observed in four surveyed fields, 57 ha in total, and disease incidence ranged from 21 to 56%. Five symptomatic plants with leaf chlorosis and root rot were randomly collected from each field for pathogen isolation. Tissue pieces from diseased roots were surface sterilized in 75% ethanol for 30 s then rinsed with sterile distilled water three times, air dried, and placed onto potato dextrose agar (PDA) medium. Five isolates, SL1, SL2, SL3, LN and KC, were purified by single-spore culturing. On PDA, colonies grew at a rate of 2-5 mm/day and produced abundant cottony, white to pink aerial mycelia and rose pigment on the reverse side of the culture plate. From 7-day-old cultures grown on carnation leaf agar (CLA), macroconidia were straight to subarcuate, with blunt and slightly hooked apical and basal cells, had three to four septa, measured 23.4 to 44.6×3.5 to 4.2 μm (n=30). Cylindrical, napiform or oval microconidia were one to two-celled, measuring 6.3 to 22.9×2.2 to 4.9 μm (n=30). Spherical chlamydospores were intercalary or terminal, in chains. Such characteristics resembled those of the species comples (FTSC; Batra and Lichtwardt 1962; Leslie and Summerell 2006). To confirm the morphological diagnosis, the genomic DNA of the isolates were extracted, the translation elongation factor 1-alpha ( ), RNA polymerase I largest subunit ( ) and second largest subunit ( ) genes were amplified with primers , and respectively (O'Donnell et al. 2010), and sequenced. Maximum likelihood analysis was carried out using MEGA 7. Sequences were 97.55% to 100% identical to corresponding DNA sequences of FTSC based on GenBank and Fusarium MLST BLASTn analysis, and deposited in GenBank (ON637268.1-ON637272.1, ON637275.1-ON637279.1, ON637282.1-ON637286.1). Based on the morphological characteristics and phylogenetic analysis, the isolates were identified as (SL1, SL2, SL3 and LN; Senatore et al. 2021) and (KC; Moreira et al. 2019). Koch's postulates were conducted to verify the pathogenicity of individual isolates. The four-leaf stage healthy tobacco seedlings (Yunyan99, n=30) were inoculated by pouring 20 mL conidial suspension (1×10 con