First Report of Diaporthe cercidis Causing Leaf Blotch of Acer pictum subsp. mono in China.

Abstract

Acer pictum subsp. mono (Maxim.) H. Ohashi is a common deciduous tree species that is widely distributed in Northeast and Northern China, including all provinces of the Yangtze River Basin (Liu et al. 2014). A foliar disease, with an incidence of ~90% (19/21 trees), occurred on A. pictum subsp. mono in a community park, Nanjing, Jiangsu, China in July 2019. On average, ~80% of the leaves per individual tree were infected by this disease. The symptoms initially appeared as brown, necrotic lesions at leaf tips, and half the leaf would become dark brown with time, and finally almost all of leaves were infected. Small pieces of leaf tissue (3 to 5 mm2) cut from the lesion margins were surface-sterilized in 75% ethanol for 30 s and 1% NaClO for 90 s, rinsed with sterile H2O three times, and placed on potato dextrose agar (PDA) at 25°C in the dark. The same fungus was isolated from 92% of the samples. The pure cultures were obtained by single-spore isolation. Three representative isolates (WJF1, WJF3 and WJF4) were obtained, and WJF1 was deposited in China's Forestry Culture Collection Center (CFCC 54806), and WJF3 and WJF4 were deposited at the Nanjing Forestry University (NFU 083 and NFU 084). The culture on PDA was white, with white vigorous aerial mycelia at the edge. Black pycnidia developed on the alfalfa stems at 25°C under a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, septate, straight, 16.4-34.7 × 1.5-3.0 μm (n = 30). Conidiogenous cells were 9.0-24.6 × 1.3-2.3 μm (n = 30). Alpha conidia were 7.0 ± 0.6 × 2.2 ± 0.2 μm (n = 30), fusiform, hyaline, smooth and multi-guttulate. Beta conidia were 25.5 ± 4.3 × 1.3 ± 0.1 μm (n = 30), hyaline, smooth and hamate. Morphological characters of all three isolates matched those of Diaporthe spp. (Gomes et al. 2013). DNA of three isolates were extracted and the internal transcribed spacer region (ITS), partial sequences of elongation factor 1-alpha (EF1-α), calmodulin (CAL), beta-tubulin (β-tub) and histone H3 (HIS) genes were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and CAL228F/CAL737R (Carbone et al. 1999), βt2a/βt2b and CYLH3F/H3-1b (Glass and Donaldson, 1995, Crous et al. 2004), respectively. The sequences of WJF1, WJF3 and WJF4 were deposited in GenBank (WJF1: Accession Nos. MW301339 for ITS, MW363932 to MW363935 for EF1-α, β-tub, HIS, and CAL; WJF3: MW453062 and MW561566 to MW561569; WJF4: MW453063 and MW561570 to MW561573). BLAST results showed that the ITS, EF1-α, β-tub, HIS, and CAL sequences of WJF1 were similar with sequences of Phomopsis liquidambari C.Q. Chang, Z.D. Jiang & P.K. Chi JQ676191 (identity = 540/540; 100%), D. huangshanensis H. Zhou & C.L. Hou MN224671 (identity = 291/292; 99%), D. pescicola Dissan., J.Y. Yan, Xing H. Li & K.D. Hyde MK691230 (identity = 438/438; 100%), D. spinosa Y.S. Guo & G.P. Wang MK726170 (identity = 437/438; 99%), D. cercidis C.M. Tian & Qin Yang MK691114 (identity = 452/452; 100%), respectively. BLAST results of WJF3 and WJF4 are list in Table 1. A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed WJF1, WJF3 and WJF4 in the clade of D. cercidis. Based on the five-locus phylogeny and morphology, WJF1, WJF3 and WJF4 were identified as D. cercidis. The pathogenicity of three isolates were tested on potted 3-yr-old seedlings of A. pictum subsp. mono, grown in a greenhouse. Healthy leaves were wounded with a sterile needle and then inoculated with 10 μL of conidial suspensions (106 conidia/mL). Control leaves were treated with sterilized H2O. A total of twelve seedlings were used for the tests, 3 seedlings per treatment, and five leaves were inoculated per seedling. Each plant was covered with a plastic bag after inoculation and sterilized H2O was sprayed into the bag twice/day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. In 5 days, all the inoculated leaves had lesions similar to those observed in the field. D. cercidis was reisolated from the lesions of the inoculated leaves and was confirmed based on morphological characteristics and ITS sequence analysis. No symptoms were observed on the control leaves, and no fungus was isolated from them. D. cercidis was previously reported on twigs of Cercis chinensis (Yang et al. 2018) and causing pear shoot canker (Guo et al. 2020). This is the first report of D. cercidis causing leaf blotch on A. pictum subsp. mono. Identification of the pathogen is imperative for diagnosing and controlling this potentially high risk disease on A. pictum subsp. mono and also for the future studies.