Identification of <i>Fusarium proliferatum</i> Causing Leaf Blight of Arecanut ( <i>Areca catechu</i> ) in China

Arecanut (<i>Areca catechu</i> L.), commonly called betel nut, is an economically important crop in China's tropical and subtropical regions. During 2023-24, 61.6%, 64%, and 72% disease incidence were observed at three locations in Wenchang county, Hainan Province, China. The characteristic symptoms were yellow to brown lesions on the older leaves, surrounded by a yellow halo circular or irregular, gradually covering the larger area, resulting in discoloration, rosish, wilting, and splitting of older leaves. Infected leaf samples were trimmed into small sections (3-5 mm), and the isolation was done on PDA, incubated at 25 °C for 7 days in the dark (Alberti et al., 2018). Five isolates (blyf1, blyf2, blyf3, blyf4, and blyf5) were purified using a single-spore isolation technique. The morphological characteristics of these isolates were compared with type isolates on PDA and carnation leaf agar (CLA) (Leslie and Summerell, 2006). The colonies on PDA were typically white or off-white on the upper surface and light yellow on the back surface. Key morphological characteristics on CLA showed microconidia in chains, ovoid and obo-void, aseptate, oval, or clavate in shape, and having length of 4.73-9.83 µm to 5.01-9.97 µm, and width of 1.30-3.88 µm to 1.90-4.08 µm, and macroconidia were long, slender, straight or slightly curved with 2-5 septa, and having length of 20.86-40.37 µm to 30.41-42.89 µm, and width of 2.12-4.65 to 2.84-4.96 µm. Conidiophores were solitary or grouped, and phialides (mono-phialidic or polyphialidic) were cylindrical to flask-shaped. Chlamydospores were absent. The internal transcribed spacer (ITS), β-tubulin gene (<i>TUB2</i>), translation elongation factor 1-alpha (TEF-1α), and RNA polymerase II subunit 2 (RPB2) amplified and sequenced using the universal primers ITS1/ITS4, T1/Bt2b, EF1/EF2, and RPB2-5f2/RPB2-7cr, respectively, confirmed these isolates as <i>F</i>. <i>proliferatum</i> when compared with other <i>Fusarium</i> spp. in the GenBank and FUSARIUM-ID v.3.0 databases (Torres-Cruz et al., 2022). The sequences were deposited into GenBank (https://www.ncbi.nlm.nih.gov/) with accession numbers: PV065590 to PV065594 for ITS, PV339649 to PV339643 for <i>TUB2</i>, PV339654 to PV339658 for TEF-1α, and PV524660 to PV524664 for RPB2. The results of the phylogenetic tree constructed using the Bayesian inference method based on ITS, <i>TUB2</i>, TEF-1α, and RPB2 placed all five isolates in the same clade as <i>F</i>. <i>proliferatum</i> (Alberti et al., 2018). Pathogenicity tests were carried out on five one-year healthy arecanut plants through artificial inoculation using spore suspension of 2 × 10⁶ conidia/mL of all 5 isolates. Control plants were inoculated using sterile water. The experiments were repeated twice under controlled conditions (25 °C and 90% humidity) for seven days, and similar symptoms were observed as in the field while the control plants remained healthy. The five isolates were consistently re-isolated from the inoculated arecanut plants, which confirmed Koch's postulates. This widely distributed pathogenic fungus has been reported in several crops (fruits, and vegetables) (Alberti et al., 2018; Sahouli et al., 2020; Alananbeh et al., 2021; Cer and Benlioglu, 2024). To our knowledge this is the first report of <i>F</i>. <i>proliferatum</i> as a pathogen of arecanut plants causing leaf blight disease in China.