Gray Blight Disease on Euonymus japonicas Caused by Pestalotiopsis disseminata in China.

Abstract

Euonymus japonicas is widely planted as an important landscape species throughout China. In June 2021, a serious gray blight disease was detected on E. japonicas in Henan Province (32°30'58" N, 112°19'44" E), causing severe defoliation of infected trees with a foliar disease incidence of 52 to 70% (n = 100). Gray spots initially appeared on leaves, gradually expanded into irregular white blotches with dark brown borders, eventually leading to wilting and death of the leaves. The junctions between the lesion and healthy tissue of infected leaves were cut into 3 × 3-mm pieces, surface sterilized with 1% NaClO solution for 1 min, rinsed in sterile water, and placed on PDA plates with 50 μg/ml of streptomycin. Three isolates (HY94, HY95, and HY98) were selected for subsequent experiments. The colonies reached 80-85 mm diam after 7 days at 25°C, with undulated margins, white to pale in color, with moderate aerial mycelium on the surface. Conidiomata were globose, solitary, and dark black. Conidia were ellipsoid, straight to slightly curved, 4-septate, 19 to 26.4 × 5 to 7.5 μm (n=100). The apical cell was cylindrical and hyaline, with 2 to 3 tubular apical appendages, unbranched, filiform, 2.5 to 3.5 μm in length. The basal appendage was single, unbranched, centric, 1.5 to 3 μm long. The characteristics were close to those of Pestalotiopsis spp. (Maharachchikumbura et al. 2013). The genomic DNA was extracted, and the rDNA internal transcribed spacer (ITS), the β-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified by primers ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Carbone and Kohn, 1999). Sequences were submitted to GenBank with accession numbers OL840327-OL840329(ITS), OL961454-OL961456(TUB), and OL961448-OL961450 (TEF1). BLASTn analyses of ITS, TUB, and TEF1 sequences exhibited 99.46, 99.05, and 96.53% similarity to the sequences of Pestalotiopsis disseminata strain MEAN1166 (ITS, 548/551 bp; MT374688) (Silva et al. 2020), PSH2000I-066 (TUB, 418/422 bp; DQ333575), and TAP29O082 (TEF1, 250/259 bp; AB453850), respectively in GenBank. The three isolates formed a clade with the type strains, MEAN 1166 and MAFF238347 of P. disseminata in phylogenetic trees, being clearly seperated from other Pestalotiopsis spp. Based on morphological and molecular evidence, the pathogen was identified as P. disseminata (Maharachchikumbura et al. 2011). To fulfill Koch's postulates, pathogenicity was tested with three isolates. Ten healthy leaves of 5-year-old intact plants were used per isolate and inoculated with mycelial plugs on both nonwounded and wounded leaves. Control leaves were inoculated with agar plugs. The inoculated plants were placed at 28°C in a greenhouse (90% relative humidity). Distinct lesions were observed after 10 days. The pathogen reisolated was identical to that of the original cultures according to phenotype and ITS sequences. The control leaves showed no obvious symptoms. P. disseminata is known to cause disease on several important plants in China, such as Camellia japonica (Zhang et al. 2012), Pinus armandii (Hu et al. 2007), and Tripterygium wilfordii (Kumar et al. 2004). This is the first report of gray blight disease caused by P. disseminata on E. japonicas in China and worldwide. The fungal pathogen identification will provide valuable information for prevention and management of gray blight disease associated with E. japonicas.