Leaf Spot Disease on Euonymus japonicas Caused by Nigrospora oryzae in China.

Euonymus japonicas belong to the family Celastraceae and is native to Japan. This ornamental plant has been widely introduced for cultivation as a hedge plant in China. From August to October 2021, severe leaf lesions were observed on E. japonicas in Meicheng garden in Nanyang (32°59'42"N, 112°33'13"E), Henan Province, China. The disease had very wide coverage in the surveyed areas, with foliar diseases reaching 50%-69% (n=200). The early symptoms were yellow or brown specks on the leaves, mostly at the tip and margin of the leaves. Then the specks gradually expanded into round amorphous and became dark brown, eventually leading to large irregular or circular lesions and even branch necrosis. Twenty symptomatic samples were collected from several individual plants, and the junction areas between infected and healthy tissues were cut into 5×5 mm pieces. The tissues were sterilized in 75% ethanol for 30 seconds and 1% NaClO solution for 1 min, rinsed thrice in sterile water and placed on potato dextrose agar (PDA) plates supplemented with 50 µg/ml of streptomycin, incubated at 25°C for 3 days. The edges of the colony were cut and transferred to new PDA plates for purification. These strains showed similar phenotypes in morphological characters. Three representative purified strains (HY12, HY16, and HY17) were used for further study. Colonies were fast-growing, massive sparse aerial hyphae, initially white, later turning gray and black. Hyphae were branched, septa, and transparent. Conidia were single-celled, dark black, oblate, or nearly spherical, and measured 10.7 to 15.4 μm × 9.8 to 15.5 μm in diameter (n=100). For molecular identification, the rDNA internal transcribed spacer (ITS), the β-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified from genomic DNA by primers ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Carbone and Kohn, 1999). Sequences were submitted to GenBank with accession numbers OL840319, OL840320, OL840321 for the ITS sequences, OL961451, OL961452, OL961453 for the TUB sequences, and OL961445, OL961446, OL961447 for the TEF1 sequences of the strains HY12, HY16, and HY17, respectively. BLASTn analyses of these sequences exhibited 99 to 100% identity to Nigrospora oryzae strains 62L1, LC6923, and DP-J2 (MZ151384 of ITS, KY019581 of TUB, and MW562242 of TEF1). These morphological features and molecular identification indicated that the pathogen possessed identical characteristics as N. oryzae (Berk. &Broome) Petch. Pathogenicity was tested through in vivo experiments. Mycelial plugs of the pathogen strains were inoculated on the wounded leaflets, meanwhile, agar plugs served as blank controls. Five 2-year-old plants were grown in pots in a climate incubator maintained at a temperature of 28°C and relative humidity of approximately 90%. Symptoms consistent with those previously described were observed on the inoculated leaves of four plants after 3 to 7 days while the control plants remained healthy. The strains of N. oryzae were reisolated from the symptomatic inoculated leaves, fulfilling Koch's postulates. N. oryzae is known to cause disease on a variety of ornamental plants in China, such as purple blow maple (Sun et al. 2011), cleyera (Wang et al. 2017), cotton rose (Han et al. 2021), and Costus speciosus (Sun et al. 2021). To our knowledge, this is the first report of N. oryzae leaf spot on E. japonicas in China. This identification research will be helpful for subsequent disease control and field management of hedge plants.