First Report of Fusarium curvatum Causing Root Rot on Huangqi in China

Huangqi (Astragalus membranaceus) is a widely cultivated traditional Chinese medicinal (TCM) herbal plant in northern and northwestern China, and, has been listed one of the "Top 10 Gansu TCMs" because of its important medicinal, edible and ecological value. The plantation scale in China reached 120,000 hm 2 in 2023. The main medicinal part of Huangqi is its root, however, root diseases negatively affect the yield and quality of the herbal plant. In June 2023 and July 2024, root rot of Huangqi was present in Shouyang town (35°07′92′′N, 104°40′09′′E), Longxi county, Dingxi city of Gansu province. Diseased plants showed stunted, slow growing, yellowing above-ground part and gradual death. Taproots were reduced in size, light brown to brown in color and decayed, and feeder roots were almost invisible. Fungi-like organisms were occasionally found on diseased roots. We used EX30 microscope (SOPTOP, China) to check diseased roots and found the presence of fungal hyphae. Meanwhile, we investigated the disease frequency and severity, and found that the 2 indexes were ~5% and severe, respectively. To isolate the pathogen of this disease, roots of diseased plants were sterilized with 75% alcohol for 30 s, washed 3 times with sterile distilled water for 1 min, and then placed on potato dextrose agar (PDA) medium and incubated at 25°C in dark. After 2-day incubation, hyphal tips from the edges of growing colonies were picked carefully and transferred to fresh PDA plates for purification. We finally obtained 15 fungal isolates with similar colony characteristics, ITS sequence and preliminary pathogenicity of which LXYPY-HQ-001, a representative isolate, was used for further identification. The colony on PDA is white (front), whereas light red in the center of the back, with abundant aerial hyphae. Growth rate was measured at ~0.92 cm/day. The colony on the back of oatmeal agar (OA) medium is purple, and the hyphae are sparse on synthetic low nutrient agar (SNA) medium. Microconidia and chlamydospores formed on the 3 media mentioned above after 14-day incubation at 25°C in dark. While, macroconidia formed only on OA medium after 14-day incubation at 25°C in dark. The microconidia were light green, without septate, nearly elliptical, and measured 5.4 (2.5 to 10.8) × 11.9 (5.9 to 21.5) µm (n = 80). The chlamydospores were grayish brown, nearly round, unicellular or multicellular, 11.6 (8.3 to 21.3) × 35.9 (15.3 to 59.0) µm in size (n = 10). Macroconidia were light green, nearly oval, rod-shaped or irregular, with 0 to 2 septate, and measured 5.5 (3.6 to 7.5) × 18.9 (11.8 to 33.5) µm (n = 30). For molecular identification, genomic DNA was extracted from mycelia using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, China). The translation elongation factor 1-alpha (tef1a), beta-tubulin (tub2) and DNA-dependent RNA polymerase II second largest subunit (RPB2) genes of the isolate were PCR amplified using primers EF1/ EF2 (O’Donnell et al. 1998), T1/ T22 (O’Donnell et al. 1997), and RPB2-5F2/ fRPB2-7cR (Sung et al. 2007, Liu et al. 1999), respectively. These amplified products were sequenced and the data were deposited in GenBank (tef1a accession PP915577, tub2 accession PP915578 and RPB2 accession PP968978). The tef1a, RPB2 and tub2 shared 99.19% (accession MH484984.1), 100% (accession MH484893.1) and 99.82% (accession MH485075.1) identity with corresponding genes of Fusarium curvatum CBS 238.94, respectively. F. curvatum belongs to the F. incarnatum-equiseti species complex (FIESC), a group of closely related species that are often difficult to distinguish based on morphology alone, thus requiring multi-gene phylogenetic analysis for accurate identification. Consistent with this, phylogenetic analysis of the concatenated tef1a, RPB2 and tub2 gene sequences using maximum likelihood method showed that LXYPY-HQ-001 clustered closely with F. curvatum CBS 238.94. Therefore, we identified the isolates in this study as F. curvatum (Zhang et al. 2024, Lombard et al. 2019) based on both morphological and molecular characteristics. Pathogenicity tests were conducted on both detached roots in petri dishes and whole plants in the field. Conidia suspensions (2×10 6 conidia/mL) were transferred to roots wounded with sterilized syringe needles, and sterile gauze was covered onto wounded parts of roots to keep wet. Sterile water was used as negative control. Petri dishes containing roots were incubated at 25°C with 75% relative humidity. All roots with conidia suspensions showed similar symptoms after 4-day incubation in petri dishes to those in the original field, while the negative control remained healthy. For pathogenicity test in the field, healthy roots of Huangqi seedlings were immersed in conidia suspensions for 30 min prior to transplanting into the field on March 28 th , 2024. Control plants were treated with sterile water. Seedlings emerged in late April, and plants were monitored regularly thereafter. Initial symptoms including root discoloration and leaf wilting were observed in mid-May, and symptoms gradually developed over time. By early June, the disease incidence (calculated as the percentage of symptomatic plants relative to the total number of inoculated plants) reached ~10% under field conditions. No symptoms were observed on control plants. Same isolates identified by above methods were reisolated from roots with symptoms, fulfilling Koch's postulates. F. curvatum was reported to cause grass pea yellowing and wilting in Iran (Soufiani et al. 2025). To our knowledge, this is the first report of F. curvatum causing root rot on Huangqi in China. Our work will be useful for the further research of this root disease and its management.