Morphology, Molecular Phylogeny, and Pathogenicity of Neofusicoccum parvum, Associated with Leaf Spot Disease of a New Host, the Japanese Bay Tree (Machilus thunbergii)

Abstract

During a survey of diseased plants on Wando Island, Korea from May to June 2020, a severe leaf spot disease was observed in the upper leaves of Japanese bay tree (Machilus thunbergii). Early symptoms were light blackish spots on the leaf surface and enlargement of older spots. Dry leaf spots surrounded with deep black margins were common throughout the plants. Symptomatic leaf samples were collected, and the causal pathogen was isolated on potato dextrose agar (PDA). Three fungal isolates (CMML20-1, CMML20-3, and CMML20-4) were cultured on PDA for morphological characterization at 25 °C in the darkness. Fungal colonies were circular, fast-growing, olivaceous to dark grey, and with abundant aerial mycelium. Sporulation was induced in 14 h-10 h light-dark conditions, and the conidia were single-celled, thin-walled with a smooth surface, ellipsoid with round apices, and measuring 17.5–20.5 (avg. 17.5) μm × 7.5–10.0 (7.9) μm. The morphological characteristics resembled those typical for Neofusicoccum parvum. Molecular identification was confirmed by partially sequencing the internal transcribed spacer (ITS) region and the translation elongation factor 1-α (EF1-α) genes. Pathogenicity tests were conducted on detached leaves and whole plants of M. thunbergii. High disease prevalence was observed, and Koch postulates were fulfilled. This is the first worldwide report of N. parvum causing leaf spots on Machilus thunbergii.