First Report of Causing Leaf Blotch of in China.

Osmanthus fragrans Lour. is widely distributed in China, Japan, Thailand and India (Zang et al., 2003) and one of the top 10 most well-known flowering plants in China. Since February, 2017, a foliar disease, with a disease incidence of ~60%, occurred on O. fragrans in a community park in Luzhai, Guangxi, China. Symptoms began as round or irregular small yellow spots and became pale brown to gray-brown with time. Small leaf tissues (3 to 4 mm2) cut from lesion margins were surface-sterilized in 75% ethanol for 30 s and 1% NaClO for 90 s before they were rinsed in ddH2O and dried on sterilized filter paper. After drying, the sterilized tissues were plated on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Five single-spore isolates were obtained and a representative isolate (GH3) was selected and deposited in the China's Forestry Culture Collection Center. The colony on PDA was white with concentric zonation and white aerial mycelia, but the reverse was yellow. Black pycnidia developed on alfalfa extract + Czapek at 25°C with a 14/10 h light/dark cycle after 17 days. Conidiophores were hyaline, branched, septate, straight to sinuous, 12.4-24 × 1.9-2.5 μm (n = 20). The conidia were fusoid, hyaline, smooth, mostly 2-guttules and measured 7.2 ± 0.7 × 2.3 ± 0.2 μm (n = 50). The morphological characters of pycnidia, conidiophores and conidia of all five isolates matched those of Diaporthe spp. (Gomes et al. 2013). DNA of isolates GH3, GH7 and GH8 was 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 GH3, GH7 and GH8 were deposited in GenBank (GH3: Accession nos. MT499213 for ITS, MT506473 to MT506476 for EF1-α, β-tub, HIS, and CAL; GH7: MT856374 and MT860397 to MT860400; GH8: MT856375 and MT860401 to MT860404). BLAST results showed that the ITS, EF1-α, β-tub, HIS, and CAL sequences of GH3 were highly similar with sequences of Phomopsis sp. [LC168784 (ITS), Identities = 506/506(100%)], Diaporthe fusicola [MK654863 (EF1-α), Identities = 274/275(99%)], D. amygdali [MK570513 (β-tub), Identities = 461/461(100%)], D. fusicola [MK726253 (HIS), Identities = 403/403(100%)] and D. amygdali [KC343263 (CAL), Identities = 428/428(100%)], respectively. 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 isolates GH3, GH7 and GH8 in the D. fusicola cluster and separated them from D. eres and D. osmanthi, which were previously reported from Osmanthus spp. (Gomes et al., 2013; Long et al., 2019). Based on the multi-gene phylogeny and morphology, all three isolates were identified as D. fusicola. The pathogenicity of GH3 was tested on 1-yr-old seedlings of O. fragrans. Healthy leaves were wounded with a sterile needle and then inoculated with either 5-mm mycelial plugs cut from the edge of a 5-day-old culture of GH3 or 10 μL of conidial suspensions (106 conidia/mL). Control leaves were treated with PDA plugs or ddH2O. Three plants were used for each treatment. The plants were covered with a plastic bag after inoculation and sterilized H2O was sprayed into the bags twice/day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. Lesions appeared 3 days later. No lesions were observed on control leaves. The same fungus was re-isolated from lesions. This is the first report of D. fusicola causing leaf blotch on O. fragrans. These results form the basis for developing effective strategies for monitoring and managing this potential high-risk disease.