First Report of Tobacco Root Rot Caused by Fusarium falciforme in China.

Abstract

China is the largest producer of tobacco (Nicotiana tabacum L.) in the world with an estimated production of 2.4 million ton per year (Berbeć and Matyka 2020). In June 2021, a root disease was observed on tobacco in three surveyed counties (Xiangcheng, Linying and Jiaxian) in central Henan. Diseased plants exhibited leaf chlorosis and brown to purplish vascular discoloration of the taproot and lateral roots. Approximately 10 to 15% of the plants were symptomatic in the nine fields surveyed, representing 60 ha in total. Root segments (0.5 to 1 cm) from ten diseased plants were surface sterilized in 75% ethanol for 30 s followed by rinsing with sterile distilled water three times. Thirty air dried root pieces were placed on potato dextrose agar (PDA) and incubated at 25℃ in the dark for 2 days. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing (Yz01 to Yz10). Colonies on PDA showed abundant white to cream aerial mycelia with a yellowish-brown center on the reverse side after 7 days, and an average growth rate of 5 mm/day. From 7-day-old cultures grown on carnation leaf agar (CLA), macroconidia had three to four septa, were falciform, with blunt apical cells and slightly hooked basal cell, and measured 20 to 41×3-6.5 μm (n=50). Spherical conidia clusters were formed at the apex of the conidiophores. Abundant reniform and cylindrical microconidia were one to two-celled, with apexes rounded, measuring 7 to 15×2 to 5 μm (n=50). The roughly spherical chlamydospores were intercalary or terminal, single or in chains, and rough walled. Such characteristics were consistent with the Fuarium solani species complex (FSSC) (Leslie and Summerell 2006). The translation elongation factor 1-alpha (EF1-α) gene of the ten cultures was amplified with primers EF1/EF2 (O'Donnell et al. 1998), and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequences of the ten cultures (Kumar et al. 2016). The RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes of the cultures were amplified with primers F5/G2R and RPB2F/R respectively (O'Donnell et al. 1998, 2010), and sequenced. The EF1-α, RPB1 and RPB2 sequences (GenBank accession nos. ON186742.1-ON186751.1, ON241133.1-ON241148.1, ON324054.1-ON324057.1) were 99.4 to 100% identical to the corresponding DNA sequences of Fusarium falciforme based on FUSARIUM-ID BLASTn analysis. Morphological and molecular results confirmed this species as F. falcifome (Díaz-Nájera et al. 2021; Velarde-Félix et al. 2022). Pathogenicity tests were performed in tobacco seedlings grown on autoclaved soil. Healthy six-leaf stage tobacco seedlings (n=30; Zhongyan 100) were inoculated by placing 7-days old wheat seed (15 seeds per plant) infested with the representative culture Yz07 around the root. Thirty seedlings inoculated with sterile wheat seeds served as controls. All the plants were maintained in a growth chamber at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Typical symptoms of foliage chlorosis and root browning were observed 7 to 14 days after inoculation for all the 90 inoculated seedlings. Fifteen diseased seedlings were randomly selected for tissue isolation, and F. falciforme was reisolated from the 15 seedlings and showed the same morphology and EF1-α gene sequence as the original isolate. Control plants remained asymptomatic and no pathogen was recovered. The results showed that F. falciforme can cause root rot of tobacco. F. falciforme was reported to cause tobacco wilt and root rot in Northwestern Argentina (Berruezo et al. 2018); however, this is the first report of F. falciforme causing root rot of tobacco in China. This species was previously reported in China affecting Weigela florida (Shen et al. 2019) and Dioscorea polystachya (Zhang et al. 2020), showing that F. falciforme has a broad host range in this country. These results may inform control tobacco root rot through improve crop rotations. Funding: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Outstanding Youth Science and Technology Fund Project of Henan Academy of Agricultural Sciences (2022YQ09) and Science and Technology Innovation Team project of Henan Academy of Agricultural Sciences (2022TD26).