First Report of Agroathelia (syn. Sclerotium ) rolfsii Causing Southern Blight on Knoxia roxburghii in Yunnan Province of China

Knoxia roxburghii (Sprengel) M. A. Rau (syn. Knoxia valerianoides Thorel ex Pitard), commonly known as Hong Daji, is a perennial herb belonging to the Rubiaceae family, with traditional medicinal uses in the treatment of cancers, carbuncles, and ascites. It is mainly cultivated in Yunnan and Guangxi Provinces, China (Chen et al. 2022). In July 2025, wilting symptoms were observed on K. roxburghii plants in a commercial plantation in Zhongshan Town, Chuxiong, Yunnan, China. (24.91°N, 101.11°E). Field surveys indicated that approximately 10% of plants were infected across the whole field, and 100% infection occurred in some individual plots. Disease symptoms first manifested as moderate to severe plant wilting and leaf chlorosis, followed by rapid collapse of roots and stems. Abundant white mycelium and brown sclerotia were primarily observed on the roots and basal stems of K. roxburghii. The sclerotia were surface-disinfected with 5% sodium hypochlorite for 3 min, rinsed three times with sterile distilled water, air-dried, placed on potato dextrose agar (PDA), incubated at 25°C in the dark. Pure cultures were obtained by transferring single hyphal tips to fresh PDA. The purified isolate was designated HDJ-BJ-1 and deposited in the China Center for Type Culture Collection (CCTCC), with the strain accession CCTCC AF 2026011. Colonies were white and cottony, often forming mycelial fans. Radial mycelial growth reached 15 mm/day on PDA, and numerous spherical sclerotia formed after 14 days. Mature sclerotia ranged from 1.15 to 2.00 mm in diameter (mean ± SD = 1.48 ± 0.16 mm, n = 50). The nuclear ribosomal internal transcribed spacer (ITS), the large subunit ribosomal RNA (LSU) region, the translation elongation factor 1-alpha (TEF1-α),and the second largest subunit of RNA polymerase II (RBP2) region were amplified with primer pairs ITS1/ITS4 (White et al. 1990), LR0R/LR5 (Xu et al. 2010), EF595F/EF1160R (Wendland and Kothe 1997), and bSr-1F/b11R1 (Remesal et al. 2013), respectively. Sequences were deposited in GenBank with accession numbers PX901757 (ITS), PX923131 (LSU), PX926091 (TEF1-α), PZ667705 (RPB2). BLAST analysis showed that the ITS sequence exhibited 100% identity with Agroathelia rolfsii (Amylocorticiaceae, anam. Sclerotium rolfsii, GenBank accession MN610000), while the LSU, TEF1-α and RPB2 sequences shared 99% identity with A. rolfsii GenBank accessions PP549543, JF267794 and OR192511, respectively. Phylogenetic relationships were inferred using the maximum likelihood method. Based on the disease symptoms, cultural characteristics, and phylogenetic analyses of ITS and LSU sequences(Wei et al. 2022; Lee et al. 2024; Lu et al. 2025), the pathogen was identified as Agroathelia rolfsii (Sacc.) Redhead & Mullineux. Pathogenicity was tested on healthy K. roxburghii seedlings in plastic pots filled with sterilized field soil. Ten plants were inoculated by placing two mycelial plugs (from 10-day-old cultures of isolate HDJ-BJ-1) per plant on the soil surface immediately adjacent to the stem base. An equal number of non-inoculated plants served as controls. All pots were individually covered with transparent polyethylene bags for 7 days to maintain high humidity and incubated in a growth chamber at 25°C with a 12-h photoperiod. Inoculated plants began to wilt at 10 days post-inoculation (dpi). By 14 dpi, 100% of inoculated plants displayed symptoms identical to those observed in the field, whereas the control plants remained symptomless. The pathogen was reisolated from the diseased roots and confirmed as A. rolfsii based on colony morphology and sequencing of ITS, LSU, TEF1-α and RPB2 regions. A. rolfsii can infect more than 500 species of plants (Daunde et al. 2020). To the best of our knowledge, this is the first report to identify K. roxburghii as a host of A. rolfsii.