First Report of Root Rot Caused by <i>Epicoccum latusicollum</i> on <i>Atractylodes macrocephala</i> Koidz. in China

Atractylodes macrocephala Koidz. (AMK) is a perennial herb whose dried rhizomes are used as a traditional Chinese medicine (Ying et al. 2026). In May 2023, root rot was first observed on AMK in Qianshan County, Anhui Province (30°50′10′′N, 116°37′50′′E), with an incidence of 30% in a 3-ha field. Diseased plants exhibited brown, water-soaked rot on both roots and rhizomes, along with leaf yellowing and wilting. For pathogen isolation, five diseased plants were randomly sampled, and each rhizome was cut into 10 pieces (5 × 5 mm). Each piece was disinfected with 75% ethanol for 1 min and 0.5% NaClO for 2 min, rinsed three times with sterile water, and incubated on potato dextrose agar (PDA) at 28°C for 7 days. Ten isolates were obtained via mycelial tip purification. Three of them exhibited similar colony morphology and were tested for pathogenicity on detached AMK roots using mycelial plug inoculation. Isolate Qsrd8 resulted in the largest lesion area (28.83 ± 3.26 mm²) among the three strains and was selected for further study. After 14 days of incubation, the PDA colonies were white-villous with a yellow reverse and concentric rings; the malt extract agar (MEA) colonies were densely white with a deep orange-brown reverse; and the oatmeal agar (OA) and pine needle agar (PNA) colonies produced superficial and immersed pycnidia. The NaOH spot test on the MEA showed a greenish reaction zone with a light brown halo. Pycnidia were dark brown to black, had a layered wall structure (with conidia produced endogenously), and were predominantly subspherical (118–150 × 91–130 μm, n = 20). The hyphae were hyaline and septate; the chlamydospores (9–27 μm in diameter, n = 20) were thick-walled, spherical to subspherical, and verrucose; and the conidia (4.2–6.1 × 1.1–2.3 μm, n = 50) were unicellular, smooth, hyaline, and elliptical to oblong. These morphological characteristics are similar to those of Epicoccum latusicollum (Chen et al. 2017; Xu et al. 2022). For molecular identification, the internal transcribed spacer (ITS), β-tubulin (TUB), and RNA polymerase II second subunit (RPB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (Mohammed and Khalil 2025), Bt2a/Bt2b (Liu et al. 1999), and RPB2-5F/RPB2-7cR (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank with the accession numbers PQ538606 (ITS), PX597401 (TUB), and PX584309 (RPB2). BLAST analysis revealed that the sequence identity of Qsrd8 was 99.4% to 100% that of the E. latusicollum strain SH1: ITS (PX929015; 523/523 nt), TUB (PX942535, 348/350 nt), and RPB2 (PX942536, 901/901 nt). A maximum likelihood phylogenetic tree based on concatenated sequences revealed that Qsrd8 clustered closely with three strains of E. latusicollum, including the ex-type isolate E. latusicollum CGMCC 3.18346 (Chen et al. 2017). Thus, both the morphological and molecular data support the identification of Qsrd8 as E. latusicollum. For pathogenicity testing, three healthy potted AMK plants (propagated from one-year-old rhizomes and grown for 50 days) were inoculated by first wounding the rhizome (approximately 3 cm below the soil surface) with a sterile syringe needle, followed by soil drenching with 10 mL of Qsrd8 conidial suspension (1 × 10⁸ conidia/mL) per plant. Three other plants were treated with sterile water using the same method to serve as negative controls. Each treatment was repeated 3 times, and all the plants were incubated in a greenhouse at 28℃. Thirty days after inoculation, the plants developed root rot and aboveground symptoms (leaf yellowing and wilting) identical to those observed in the field, whereas the control plants remained asymptomatic. No E. latusicollum were isolated from the surface-sterilized root tissues of the control plants. Three Epicoccum strains were reisolated from the diseased roots of the inoculated plants, and the isolate CFL2 was successfully confirmed as Epicoccum on the basis of its morphological features and ITS sequence, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of E. latusicollum causing AMK root rot in China, providing a basis for targeted disease management.