First report of a leaf spot disease of Acer truncatum by Neofusicoccum parvum in Jiangsu Province of China.

Abstract

Acer truncatum, originating in China, is an ornamental tree species with high economic value. In September 2022, a new leaf spot disease was observed on seedlings of A. truncatum in a tree nursery from the Jiangsu Academy of Forestry (118°45'517.30″E, 24 31°51'27.94″N) in China. According to statistics, 62.5% of 1600 A. truncatum seedlings suffered from the disease. The first symptoms were black to brown spots appearing on the infected leaves. Subsequently, the spots gradually expanded into larger areas, and finally the entire leaf turned yellow and fell off. The diseased leaves were collected and sections of 3 to 4 mm were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 sec. and 1.5% NaClO for 90 sec., rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in darkness. Pure cultures were obtained by monosporic isolation. Six fungal cultures with similar morphological characteristics were obtained from the infected tissues, and they accounted for 70% of the total isolates. Colonies were initially white but turned dark gray following 7 days of incubation at 25°C in the dark. To induce sporulation, colonies were grown on 2% water agar and incubated under UVA light at 25°C for two weeks. The conidiophores produced conidia, and conidia were hyaline, unicellular, nonseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and were 15.3 to 20.4 × 4.5 to 7.4 μm (n=35, counted from the two selected isolates). These characteristics were consistent with the description of Neofusicoccum sp. (Kirk et al. 2008). Isolates YBF2-1 and YBF5-1 were selected as representative for molecular identification. The internal transcribed spacer region (ITS), the translation elongation factor 1-alpha gene (EF1-α), and the beta-tubulin gene (TUB2), using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995) were amplified, respectively. The obtained ITS (GenBank Accession No. PP980685, PQ222752), EF1-α (No. PQ009204, PQ227810), and TUB2 sequences (No. PP993456, PQ227811) all showed >99% homology with several GenBank sequences of Neofusicoccum parvum (MK370685, KC818612 for ITS; MH118932, KJ126847 for EF1-α; and MN318109, OL456719 for TUB2, respectively). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The two isolates were identified as N. parvum. To test pathogenicity, 15 leaves on three one-month-old A. truncatum seedlings (five leaves from each seedling) were wounded with a sterile needle inoculated with 20 μL conidia suspension (1×106 spores/mL) on the left sides of the leaves, using isolate YBF2-1, while the same size droplet of sterilized water was used on the right sides of leaves. Each plant was separately covered with clear polyethylene bags to maintain about 80% relative humidity at 25℃. After 5 days of inoculation, typical symptoms were found on the left sides of treated leaves, and no symptoms occurred on the right sides of the leaves (the control). The inoculation experiment was conducted three times. Subsequently, the same fungus was reisolated and identified from six leaves of three inoculated seedlings. This is the first report of N. parvum on A. truncatum in the world, and it provides an important reference for the biology and epidemiology of N. parvum.