First Report of Fusarium annulatum Causing Leaf Wilt and Fruit Rot on Areca catechu in Hainan Province, China

Areca catechu, a major economic crop in Hainan Province, China, has sustained steady production growth over the past five years, reaching an annual yield of 276,200 tonnes. (Wang et al. 2023). In May 2025, symptoms of fruit rot disease were observed in a 0.667 ha. A. catechu plantation in Ledong County (18°56.4'N, 108°80.8'E), Hainan Province, China. The maximum disease severity reached 75%, with an incidence rate of 80%. Initial symptoms included visible white mycelia on the epicarp and pedicel, tissue softening, and progressive rot extending from the pedicel to the fruit base. Ten symptomatic fruits were surface-sterilized in 0.05% sodium hypochlorite for 2 min, rinsed three times with sterile distilled water, and air-dried. Sterilized fruits were placed in sterile containers and incubated at 4°C with 85% relative humidity in a refrigerator. Following mycelial development on lesions, hyphal tips were transferred to potato dextrose agar (PDA) by streaking. Initial isolates were subcultured on fresh PDA to obtain pure cultures. All ten isolates consistently formed white to grayish-white colonies. Isolate G2 initially produced white, fluffy aerial mycelia with purple submerged mycelia. Over time, aerial mycelia turned grayish-white while submerged mycelia became purplish-red. Conidia were initially elliptical, unicellular, and symmetrical; mature conidia were clavate and septate. Conidial dimensions averaged 10.26 ± 0.24 µm (width) and 29.84 ± 1.70 µm (length). Genomic DNA was extracted and the ITS, TEF1-α, and RPB2 loci of strain G2 were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R, and RPB2-5F2/RPB2-7cR, respectively (Carbone and Kohn 1999; Liu et al. 1999; Rehner and Buckley 2005). Sequences were deposited in GenBank under accession numbers ITS (PX022875), TEF1-α (PX060362), and RPB2 (PX060361). BLAST analysis revealed 98-100% identity with reference sequences of the same loci of Fusarium annulatum strain CBS:258.54. Concatenated multi-locus (ITS/TEF1-α/RPB2) phylogenetic analysis was performed and a maximum likelihood tree was constructed. To fulfill Koch’s postulates, unwounded healthy fruits of uniform size were purchased locally. Fruits were surface-sterilized in 0.05% NaOCl for 2 min. Following careful pedicel removal to create wounds, 20 µl of G2 conidial suspension (1 × 10⁷ CFU/ml) was inoculated onto each wound site. Sterile water served as a negative control (five replicates per treatment). Inoculated fruits were incubated in sterile containers at 25°C under 12-h light/dark cycles at ambient humidity. After 8 days, inoculated fruits developed dense white mycelia at the pedicel end, followed by tissue softening, progressive rot from pedicel to base, complete yellowing, and slimy epicarp formation—consistent with field symptoms. Control fruits remained asymptomatic. Re-isolated pathogens from inoculated fruits showed identical morphological characteristics to the original strain. Leaves were similarly inoculated and developed dark brown lesions after 8 days. Collectively, symptomatology, morphological characteristics, pathogenicity assays, and multi-locus phylogenetic analysis demonstrate that Fusarium annulatum is the causal agent of A. catechu fruit rot and leaf spot disease. This constitutes the first report of F. annulatum causing fruit rot and leaf blight on A. catechu in Hainan Province, China. These findings provide insights for the development of crop loss mitigation strategies.