First Report of Fusarium humuli Causing Stem Spot in Nicotiana tabacum in China

Abstract

Tobacco is one of the most important economic crops in China. Disease is one of the main factors affecting the quality of tobacco production (Cai et al. 2022). Stem spot disease of tobacco was observed in the Planting Demonstration Garden in Chang Ning (26°37N; 112° 31E), Hunan Province of China, from May to June 2023. The disease seriously retarded tobacco growth and the incidence rate was about 30-50% of the plants(Yun Yan 87). Most of infected tobacco had black spots on the stems, and the spots expanded and joined together quickly, while many stems turned black and withered. For pathogen isolation, symptomatic stem samples were collected and disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 1 minute, followed by rinsing with sterile distilled water three times. Subsequently, small pieces (5 × 5 mm) of diseased tissues were placed on potato dextrose agar (PDA) and incubated in the dark at 25 °C for 24 h to 36 h. The emerging fungal hyphal tips were transferred to PDA and purified by the single-spore method(Yu et al. 2022). In total, 16 cultures with the same appearance were isolated from 30 disease spots on the stem. Strain coded as hnxryc2 was randomly selected for identification. After culturing in PDA for 7 days, white and dense colonies wereobserved with a mean radial growth rate of 6.4 mm/day. The strain cultured 10 days on SNA. Morphological observations were made on 10-day-old culture on SNA medium, and macroconidia were sickle-shaped and slightly curved, with 3-5 septa (2.32-7.00 µm × 0.53-1.17 µm, n = 50), neither microconidia nor chlamydospores were observed. These morphological characteristics were consistent with the description of Fusarium humuli (Wang et al. 2019, Li et al. 2023). Furthermore, primers ITS1/ITS4, EF728F/EF986R, RPB1-F5/RPB1-R8 and fRPB2-5F2/fRPB2-7cR(Xie et al. 2023) were used to amplify the ITS region, EF-1α, RPB1, and RPB2 from strain hnxryc2, respectively. The sequence alignment of hnxryc2 with the NCBI database and FUSARIOID-ID shows the following results: The sequence of ITS region(GenBank accession number PP543715) was 100% identical to these of Fusarium sp. (MN428026.1), the sequences of EF-1α, RPB1, and RPB2 of strain hnxryc2(GenBank accession numbersOR257586, OR326856 and OR257587 respectively) were 99% to 100% identical to these of F. humuli (GenBank accession numbers MK289578.1, MZ824672.1 and MZ824673.1, respectively). Then a phylogenetic tree based on ITS region, EF-1α, and RPB2 sequences was constructed (Kroon et al. 2004). The strain hnxryc2 was more closely related to F. humuli (CGMCC3.19374 GenBank accession nos. MK280845.1, MK289570.1 and MK289724.1, respectively), with bootstrap values 88%. Pathogenicity tests were performed on detached stems of tobacco and potted plants. Wounded stems were inoculated with conidial suspensions (100 μL, 1×107 spores/mL), and the controls were inoculated with sterile water (Xu et al. 2023). The inoculated detached stems were kept in humid chambers (Zhong et al., 2019), each treatment was given a 12h/12h light/dark cycle at 25°C. Deep black spots were observed for 3 days after inoculation. After 9 days, typical symptoms similar to the original diseased plants in the field were found on all inoculated stems, while the control stems did not exhibit any symptoms. Pathogenicity assays were repeated thrice. The pathogen F. humuli was successfully reisolated from the stem of inoculated samples showing symptoms. To our knowledge, this is the first report of F. humuli inducing stem spot on tobacco in China. Since F. humuli is a common pathogenic fungus that infects different plant species, more attention should be paid to its prevalence in tobacco, and the potential risk of a disease outbreak in other provinces of China.