First report in China of Fusarium humuli as a causative agent of Chinese yam wilt

Abstract

Wilt is one the most serious soil-born fungal diseases of Chinese yam (Dioscorea polystachya Turczaninow cv. Tiegun), affecting plant production in many growing regions in Jiangxi province, China. The average annual incidence of wilt is 45-80%. In 2020, affected plants with wilt symptoms including withered and dried leaves, vascular discoloration, and brown necrotic stem lesions at the soil line or in the crown of the plant (Figure 1A-D) were collected from the Ruichang, Yongfeng and Taihe production areas (four fields per location) in Jiangxi province. A total of fifteen isolates were recovered from the infected stem tissues of Chinese yam and purified by single spore cultures on PDA growth medium. The fifteen isolates were similar in morphology so isolate JXRC11 was selected to be representative of the group. Pure fungal colonies of JXRC11were found to be round, white, with margin entire (Figure 1E). Macroconidia with 3-5 septations were straight to slightly curved, 23.8-40.3 µm in length and 2.6-3.9 µm in width, with predominantly 5-septate macroconidia on carnation leaf agar (CLA) (Figure 1F). However, neither microconidia or chlamydospores were observed on CLA. The morphological characteristics of the isolate were consistent with the description observed previously for Fusarium humuli species complex (Wang et al. 2019). To confirm morphological identification, ITS, CAM, TEF-1α, RPB1 and RPB2 were amplified using the primers ITS5/ITS4 (White et al. 1990), CL1/CL2A (O'Donnell et al. 2000), EF1/EF2 (O'Donnell et al. 1998), Fa/G2R (O'Donnell et al. 2010), and 5f2/11ar (O'Donnell et al. 2010), respectively. BLASTn analysis of the ITS sequence (GenBank accession no. MZ768912), EF-1α (MZ824669), CAM (MZ824670), RPB1 (MZ824672) and RPB2 (MZ824673) alignment showed 99.55%, 99.68%, 99.85%, 97.61% and 99.76% identity to those of F. humuli CQ1039 (MK280845, MK289570, MK289712, MK289840 and MK289724), respectively. Multilocus phylogenetic analyses showed that the sequences of ITS, CAM, EF-1α, RPB1, and RPB2 of the isolate belonged to the incarnatum clade (FIESC-33) of the F. incarnatum-equiseti species complex with an independent branch (Figure 2). Pathogenicity tests were conducted on one-month-old Chinese yam seedlings using a seedling root dip method (Li et al. 2013). The roots and rhizomes of seedlings grown to two meters in height were dipped into spore suspensions (1×106 spores/mL) of isolate JXRC11 for 30 min and then transferred into 20 cm diameter plastic pots containing steam-sterilized soil and placed in a greenhouse under 12 h photoperiod. After 15 d, the inoculated seedlings showed typical wilt symptoms similar to those observed in farm fields, whereas the control remained unaffected (Figure 1G-H). The pathogen was then re-isolated from the infected plants, the re-isolations were identified as F. humuli by sequencing EF-1α, fulfilling the Koch's postulates. It has been reported that the pathogen F. oxysporum Schlecht causes Fusarium wilt in five species of Dioscorea (Nwankiti and Arene, 1978). Moreover, at least 5 species of Fusarium were identified as a causative agent of Chinese yam wilt (Fang et al. 2020). To our knowledge, this is the first report of Fusarium wilt disease on Chinese yam caused by a member of the F. humuli in Jiangxi, China. This report will contribute to developing management strategies to control the disease.