Ligusticum chuanxiong (known as Chuanxiong in China) is a traditional edible-medicinal herb, which has been playing important roles in fighting against COVID-19 (Ma et al. 2020). In March 2021, we investigated stem rot of Chuanxiong in six adjacent fields (~100 ha) in Chengdu, Sichuan Province, China. The disease incidence was above 5% in each field. Symptomatic plants showed stem rot, watersoaked lesions, and blackening with white hyphae present on the stems. Twelve symptomatic Chuanxiong plants (2 plants/field) were sampled. Diseased tissues from the margins of necrotic lesions were surface sterilized in 75% ethanol for 45 s, and 2% NaClO for 5 min. Samples were then rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) at 25ºC for 72 h. Fourteen fungal cultures were isolated from 18 diseased tissues, of which eight monosporic isolates showed uniform characteristics. The eight fungal isolates showed fluffy white aerial mycelia and produced yellow pigments with age. Mung bean broth was used to induce sporulation. Macroconidia were sickle-shaped, slender, 3- to 5-septate, and averaged 50 to 70 μm in length. Based on morphological features of colonies and conidia, the isolates were tentatively identified as Fusarium spp. (Leslie and Summerell 2006). To identify the species, the partial translation elongation factor 1 alpha (TEF1-α) gene was amplified and sequenced (O’Donnell et al. 1998). TEF1-α sequences of LCSR01, LCSR02 and LCSR05 isolates (GenBank nos. MZ169386, MZ169388 and MZ169387) were 100%, 99.72% and 99.86% identical to that of F. asiaticum strain NRRL 26156, respectively. The phylogenetic tree based on TEF1-α sequences showed these isolates clustered with F. asiaticum using Neighbor-Joining algorithm. Furthermore, these isolates were identified using the specific primer pair Fg16 F/R (Nicholson et al. 1998). The results showed these isolates (GenBank nos. MZ164938, MZ164939 and MZ164940) were 100% identical to F. asiaticum NRRL 26156. Pathogenicity test of the isolate LCSR01 was conducted on Chuanxiong. After wounding Chuanxiong stalks and rhizomes with a sterile needle, the wounds were inoculated with mycelia PDA plugs. A total of 30 Chuanxiong rhizomes and stalks were inoculated with mycelia PDA plugs, and five mock-inoculated Chuanxiong rhizomes and stalks served as controls. After inoculation, the stalks and rhizomes were kept in a moist chamber at 25°C in the dark. At 8 days post inoculation (dpi), all inoculated stalks and rhizomes exhibited water-soaked and blackened lesions. At 10 dpi, the stalks turned soft and decayed, and abundant hyphae grew on the exterior of infected plants, similar to those observed in the field. No disease symptoms were observed on the control plants. The pathogen was re-isolated from the inoculated tissues and the identity was confirmed as described above. Ten fungal cultures were re-isolated from the 10 inoculated tissues, of which nine fungal cultures were F. asiaticum, fulfilling Koch’s postulates. To our knowledge, this is the first report of F. asiaticum causing stem rot of Chuanxiong in China. Chuanxiong has been cultivated in rotation with rice over multiple years. This rotation may have played a role in the increase in inoculum density in soil and stem rot epidemics in Chuanxiong. Diseased Chuanxiong may be contaminated with the mycotoxins produced by F. asciaticum, 3-acetyldeoxynivalenol or nivalenol, which may deleteriously affect human health. Therefore, crop rotations should be considered carefully to reduce disease impacts.
Diversity, Chemical Constituents, and Biological Activities of Endophytic Fungi Isolated From Ligusticum chuanxiong Hort