First Report of <i>Aspergillus flavus</i> Causing Root Rot of <i>Aucklandia lappa</i> in China

Aucklandia lappa Decne. rhizome is used in Traditional Chinese medicine for its anti-fungal, anti-oxidant and anti-cancer properties. However, continuous monoculture of A. lappa Decne., along with drought or high temperature conditions, has led to the accumulation of soil-borne pathogens, resulting in an increase in the severity of root rot over the years. In June 2024, root rot symptoms were observed in A. lappa Decne. in a 5-ha field in Weixi County, Diqing Prefecture, Yunnan Province. Typical disease symptoms included browning and rot of the stem base and vascular tissues, and the aerial parts exhibited extensive leaf yellowing, defoliation, wilting, ultimately leading to plant death (Supplementary Figure 1). Approximately 20% of the plants were affected with more severe symptoms in 2-year-old plants. Ten samples of the diseased root tissue were collected and surface-sterilised by immersion in 75% ethanol for 30 s, followed by 2% sodium hypochlorite for 2 min and then rinsed three times with sterile distilled water. Subsequently, the junction between diseased and healthy tissue was used for inoculation onto potato dextrose agar. After incubating at 25°C for 4 days, isolates from eight diseased tissue samples formed yellow–green powdery colonies resembling Aspergillus, and 12 isolates were obtained through single-spore isolation. Moreover, microscopic examination revealed that the hyphae of all the isolates were compartmentalised and bore unicellular conidiophores (2–4 µm in diameter) and spherical to subglobose conidia measuring 3.1–5.2 µm in diameter (average 4.2 ± 0.3 µm, n = 20), consistent with the morphological characteristics of Aspergillus flavus (Rodrigues, 2007). For molecular identification, genomic DNA from strain A06 was extracted to amplify the RNA polymerase II subunit gene (RPB2) and internal transcribed spacer (ITS) region using primers RPB2-5F2/RPB2-7cR and ITS1/ITS2, respectively. The RPB2 and ITS gene sequences were deposited in GenBank (ITS: PV230515, RPB2: PV477850). Sequence analysis revealed that the ITS and RPB2 of A06 exhibited 99.82% and 100.00% identity with those of A. flavus (ITS: MK087749, RPB2: MZ826464), with sequence coverage of 99% for ITS and 100% for RPB2. Phylogenetic tree analysis (Supplementary Figure 2) demonstrated that strain A06 clusters with A. flavus. A spore suspension of A06 (1 × 106 CFU/mL) was smeared on healthy roots (1-year-old) with brushes, and roots in the control group were treated with sterile water (Tang et al., 2023); six plants were treated per group. After inoculation, the plants were allowed to grow for 14 days at 26°C and 60% relative humidity. Symptoms similar to those observed under natural conditions appeared on the plants inoculated with A06 (6/6 positive), and the control plants did not show any signs of infection (0/6 positive). Moreover, the same fungal strain was re-isolated from the inoculated roots and identified as A. flavus through ITS and RPB2 sequence analysis, thereby fulfilling Koch’s postulates. A. flavus is a major fruit rot pathogen in Solanum lycopersicum (Tijjani et al., 2014) and sorghum seeds (Divakara et al., 2014). Additionally, A. flavus causes root rot in Manihot esculenta Crantz. (Ubalua et al., 2007) and groundnut (Sharma et al., 2012). To the best of our knowledge, this is the first report of root rot in A. lappa Decne. caused by A. flavus. These results provide a basis for correct diagnosis and management of root rot.