First report of <i>Fusarium oxysporum</i> wilt of <i>Angelica sinensis</i> in China

Angelica sinensis (Oliv.) Diels (Umbelliferae) is a traditional Chinese medicinal plant, which is mainly used in China to treat anemia and dysmenorrhea (Committee of China Pharmacopoeia, 2020). It is mainly cultivated in Gansu, located in the northwest of China, and the cultivation area is around 2.7 × 104 ha in 2020, accounting for more than half of the total planting area in China. In August 2021, an acute fungal wilt disease with 20%-25% disease incidence was found on A. sinensis in the fields of Yuzhong County, Lanzhou City, Gansu province. Initially, the plants appeared visibly wilted. Later, the aboveground parts of the plants showed widespread wilting and the root epidermis was shriveled in some plants. The vascular bundles of the roots turned brown after splitting the plants longitudinally (Fig. 1). In some plants with severe symptoms the pith had dried. Finally, the whole plant wilted and died. Discolored root and stem tissue of the diseased plants was cut into 3-5 mm2 segments, surface disinfected 1 min in 0.1% HgCl2, rinsed four times with sterilized water, and incubated on potato dextrose agar (PDA) at 25°C for 5 days. One fungus DGKW was isolated with a 34% isolation frequency. For the pathogenicity test, a spore suspension (1 × 106 spores/ml) was prepared by rinsing a 7-day-culture on PDA with sterilized water. A 25 mL spore suspension was poured into 15 pots around the root and basal leaf branch of six-month-old A. sinensis seedling. For the controls, another 15 pots of seedlings were tested with the same quantity of sterile distilled water as the control. The seedlings were placed in a greenhouse at 25 ± 2°C, with a relative humidity of 60% to 70% and a 12 h light/dark cycle. Wilting symptoms appeared 25 days after inoculation (Fig. 1). The pathogenic fungus was re-isolated from brown vascular bundle tissue to fulfill Koch's postulates. The pathogen was transferred to Carnation Leaf Agar (CLA), Spezieller Nährstoffarmer Agar (SNA), and PDA media for morphological identification (Summerell et al. 2006). Aerial mycelia were white with a purple-tinge, and the colony diameter at 7 days was 8.1 cm on PDA. Macroconidia were abundant on CLA and microconidia and chlamydospores were abundant on SNA. Macroconidia were falcate, the apical cells were long and tapering, and the basal cell was foot-shaped. They were 1 - 4 septate，12.0 - 21.0 μm × 3.0 - 6.0 μm for 1-septate spores, 20.1 - 32.0 μm × 3.4 - 6.0 μm for 2-septate spores, 20.5 - 35.0 μm × 3.9 - 6.0 μm for 3-septate spores, 42.5 - 45.6 μm × 5.0 - 6.0 μm for 4-septate spores. Microconidia were aseptate, 7.0 - 14.9 μm×2.0 - 4.5 μm while chlamydospores were terminal or intercalary, solitary or in pairs, 7.5 - 20.0 µm × 7.5 - 15.0 µm. For molecular identification, portions of the TEF1, RPB1, RPB2 genes were amplified and sequenced with the primers EF1/EF2, Fa/G2R, and 5f2/7cr (O'Donnell et al. 2022). The 688 bp (TEF1), 1789 bp (RPB1), and 1005 bp (RPB2) sequences were deposited in GenBank (Accession Nos. PP256492, PP256490 and PP256491). Sequences of the isolate DGKW and those of 28 other strains were concatenated to construct a maximum likelihood phylogenetic tree with Mega 11.0 (Fig. 2). Sequences of isolate DGKW were clustered with the corresponding sequences of F. oxysporum NRRL 20433 which is nested within the F. oxysporum species complex. Based on the above results, the pathogen was identified as F. oxysporum. To our knowledge, this is the first report of A. sinensis wilt caused by F. oxysporum. This finding provides a scientific basis for farmers to prevent and control Fusarium wilt of A. sinensis timely and effectively.