Plant Dis. 2022 Apr 29. doi: 10.1094/PDIS-10-21-2186-PDN. Online ahead of print.
Industrial hemp (Cannabis sativa L.) is an ancient and economically important crop used in food, medicine, textile, and paper industries (Chandra et al. 2017). In July 2021, an estimated 30% of the industrial hemp plants showed wilted leaves and root rot in the greenhouse at the Modern Agriculture Demonstration Area Management Center, Harbin City, Heilongjiang Province, China. Initially, the diseased plants exhibited green and reversible wilting of lower canopy leaves. Upon progression the plants showed irreversible wilting. The epidermal tissue of root and rhizome showed slight cracks and the vascular bundle exhibited light brown discoloration, and then died. Six randomly selected disease plants were collected. Small fragments (5 mm) were cut from the infected roots, surface-sterilized with 70% ethanol for 30s and 1% sodium hypochlorite for 5 min and rinsed three times in sterile H2O. Then the small pieces were embedded on potato dextrose agar at 25℃ for 4 days and sub-cultured by hyphal tipping to isolate the fungus. A single-spore culture was obtained by monosporic isolation. The colonies were characterized by an abundant white cottony mycelium, which became gray or purple with age. The macroconidia were transparent, short to medium in length, straight to slightly curved, septate 0 to 4, 16.8 to 26.6 µm long × 3.5 to 4.1 µm wide. The apical cells were long and tapering to a point and the basal cells were notched. Microconidia were elliptic or kidney-shaped, and septate 0 to 4. The conidia were 4.2 to 11.3 µm long × 3.5 to 5.5 µm wide (n = 50). The morphological characteristics were very similar to those of Fusarium oxysporum (Leslie and Summerell 2006). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1) and RNA polymerase II beta subunit (RPB2) genes were amplified and sequenced with the primers ITS1/ITS4, EF-1/EF-2 (Uwaremwe et al. 2020), and 5f2/7c (O’Donnell et al. 2010). The 520 bp (ITS), 948 bp (TEF1), and 861 bp (RPB2) sequences were deposited in GenBank with acce. nos. MZ722998, OK180473 and OK180474, respectively. NCBI BLAST analysis showed 98 to 100% similarity with the sequences of F. oxysporum. Moreover, the sequences alignment similarity for the six isolates were 100%. Based on the morphological and molecular characteristics, the isolates were identified as F. oxysporum. For the pathogenicity test, 20 seedlings were inoculated 30 ml of a conidial suspension (106 conidia/ml) using the root dip method. Another set of 20 seedlings were inoculated with the same quantity of sterile distilled water as the controls. After inoculation, all seedlings were maintained in a greenhouse at 25°C ± 2, with a relative humidity of 60 to 70% and a 16 h light/8 h dark cycle. This test was repeated twice. The leaves of the inoculated seedlings gradually became yellow and exhibited wilting within 15 to 20 days, the epidermal tissue of root showed light brown discoloration. Eventually the plants were dead within 40 to 50 days after inoculation. The control seedlings did not show any wilt symptoms. F. oxysporum was re-isolated from the infected root tissues to fulfill the Koch’s postulates. In addition to F. oxysporum, F. brachygibbosum, Pythium aphanidermatum, F. solani, and F. equiseti have also been reported to cause wilt symptoms of industrial hemp (Zamir et al. 2018). To our knowledge, this is the first report of Fusarium wilt on C. sativa caused by F. oxysporum in the Northeast China.
Source: ncbi 2