First report of Diaporthe acuta causing leaf blight of Acer palmatum in China.

Abstract

Acer palmatum Thunb. is an important ornamental deciduous tree with colorful foliage, and widely cultivated in Japan, Korea and China (Carlos et al. 2016). In October 2021, a foliar disease of ~95% incidence was observed on A. palmatum in three community parks, Shaoxing, Xuzhou, and Wuhan cities, China. The symptoms appeared as brown necrotic lesions at the tips, margin, and surface of leaves. Thirty leaves with symptoms from three trees were collected from the three parks. Small pieces (3 to 5 mm2) cut from the lesion margins were placed on potato dextrose agar (PDA) after surface-sterilized and incubated at 25°C in the dark, following the protocol described previously (Wan et al. 2022). The same fungus was isolated from 31% of 150 tissue pieces. Pure cultures were obtained from the tip of hyphae. Three representative isolates (WH52, SX13, and XZ96) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelia, cottony, and the reverse was white. Gray pycnidia developed on the sterile alfalfa stems at 25°C with a 14/10 h light/dark cycle in 30 days. Conidiophores were hyaline, cylindrical, septate, branched, smooth, 14.3-37.2 × 1.5-3.7 μm (n = 30). Conidiogenous cells were cylindrical, 5.6-21.6 × 1.3-2.1 μm (n = 30). Alpha conidia were aseptate, fusiform to oval, 6.5 ± 0.6 × 2.2 ± 0.2 μm (n = 50), bi- or multi-guttulate. Beta conidia were aseptate, hyaline, and curved, 31.0 ± 3.5 × 1.0 ± 0.1 μm (n = 30). Gamma conidia were aseptate, infrequent, botuliform, 12.4 ± 1.2 × 1.4 ± 0.1 μm (n = 10). Morphological characteristics of the three isolates matched those of Diaporthe spp. (Gomes et al. 2013). DNA of the three isolates was extracted and the internal transcribed spacer region (ITS), histone H3 (HIS), partial translation elongation factor 1-alpha (TEF1-α), beta-tubulin (TUB), and calmodulin (CAL) genes were amplified with primers ITS1/ITS4 (White et al. 1990), CYLH3F/H3-1b (Glass and Donaldson, 1995; Crous et al. 2004), EF1-728F/EF1-986R (Carbone et al. 1999), Bt2a/Bt2b (Glass and Donaldson 1995), and CAL-228F/CAL-737R (Carbone et al. 1999), respectively. The genomic DNA sequences were deposited in GenBank with Accession Nos. OP522005, OP522447, OP522448, and OP566419 to OP566430 (Supplementary Table 1). BLAST search of the sequences from the three isolates showed high similarities with sequences of Diaporthe acuta Y.S. Guo & G.P. Wang (ex-type PSCG 047). BLAST results were listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed WH52, SX13, and XZ96 in the clade of D. acuta. Based on the phylogeny and morphology, the three isolates were identified as D. acuta. The pathogenicity was tested on potted 3-yr-old seedlings of A. palmatum. Healthy leaves wounded with a sterile needle (1 mm in diameter) were inoculated with 5-mm plugs from the edge of 3-day-old culture of the three isolates. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags to maintain humidity in a greenhouse at the day/night temperatures at 25 ± 2°C. The plastic bags were removed on the fifth day. Five days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe acuta was reisolated from the lesions on the inoculated leaves and was confirmed based on morphological characteristics and ITS sequence analyses. No fungus was isolated from the controls. Diaporthe acuta was previously reported to cause pear shoot canker in China (Guo, et al. 2020), and D. foliicola, D. monospora and D. nanjingensis caused leaf blight of A. palmatum (Wan et al. 2022). This is the first report of D. acuta causing leaf blight of A. palmatum. This finding will provide an effective basis for developing control strategies for the disease.