First report of Diaporthe chamaeropicola causing leaf spot of Arecanut (Areca catechu L.) in China

Arecanut (Areca catechu L.), commonly known as betel nut, is one of the economically important crops in the family Arecaceae, cultivated in Hainan Province, China. This region's warm, humid climate promotes the growth of microorganisms, especially pathogenic fungi. From 2023 to 2024, a leaf spot disease was observed on arecanut seedlings in the research area of the Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences. The disease affected approximately 8% of seedlings across 6 hectares under natural environmental conditions. The main objective of this study was to identify the causal agent of this unknown disease of arecanut based on pathogenicity tests and morpho-molecular identification using phylogenetic analysis. A total of two isolates were obtained from symptomatic leaf samples collected between 2023 and 2024. The infected leaves exhibited typical disease symptoms, characterized by yellowish to brown spots on the leaf blades, which were regular to irregular in shape and surrounded by a chlorotic yellow halo. The minor spots became more prominent, reducing the chlorophyll content, causing the leaves to split from the center, and giving a broom-like appearance. The color ranged from black to off-white, and the leaf eventually dried and dropped off. We obtained monoconidial isolates by transferring pycnidia onto water agar supplemented with alfalfa rod medium at 25°C and then purified them using the single-spore technique (Shabong and Kayang 2023). The morphological characteristics of the fungal colonies were light grey, with a dense and fluffy appearance, and exhibited a small amount of yellow pigment deposition on the backside of the colony. On the water agar plus alfalfa rod medium, the pile of conidia exuded from the sporangium, appearing as black droplets. α-spores were aseptate, colorless, and transparent, oval to spindle-shaped, 3.9–9.5 × 2.1–3.0 μm; β-spores were colorless, transparent, linear, smooth, curved, base truncated,15.3–28.6 × 1.3–1.9 μm, like those of Diaporthe spp. (Udayanga et al. 2015; Moodispaw et al. 2023). Partial sequences of the internal transcribed spacer (ITS) (White et al. 1990), calmodulin (CAL) (Carbone and Kohn 1999), and translation elongation factor 1-a (TEF) (Carbone and Kohn 1999) were amplified. GenBank accession codes for BLX2 and BLX3 were PV653305 and PV653306 for ITS, PV696108 and PV696109 for CAL, and PV696110 and PV696111 for TEF, respectively. A multilocus phylogenetic analysis based on ITS, CAL, and TEF, using the neighbor-joining method, showed that the two isolates formed a distinct lineage with Diaporthe chamaeropicola CDP460. Arecanut pot plants were artificially inoculated with these isolates using a 1 × 10 6 conidia/ml suspension, while sterile distilled water mixed with Tween was used as a control. Five plants were used for each replication, and all the experiments were repeated thrice and incubated at 26±2°C, >95% RH, and a 12-h light and dark period. Leaf symptoms with brown leaf spots surrounded by regular to irregular chlorotic yellow halo were observed on the inoculated plants, whereas the control plants did not exhibit any symptoms. Morphological and molecular tests confirmed the identity of the reisolated fungal colonies, fulfilling Koch's postulates. This first report of D. chamaeropicola causing leaf spot on arecanut in China highlights a new threat to arecanut cultivation, emphasizing the need for early detection and effective disease management strategies.