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#35973084   2022/08/16 To Up

First Report of Causing Leaf Spot of var. in China.

var. (K. Koch) Zabel ex Rumpl. is an evergreen tree, used as a landscape plant in China. In June 2019, a foliar disease of ~60% incidence was observed on var. at the campus of Nanjing Forestry University, Jiangsu, China. The symptoms were initially irregular small red-brown spots, later enlarged and became brown to black. Small pieces of tissue (3 to 4 mm) cut from lesion margins were surfaced sterilized in 75% ethanol for 30 s and 1.5% NaClO for 60 s, then rinsed in sterile water and placed on potato dextrose agar (PDA) at 25℃. Pure cultures were obtained from the tip of hyphae. Using the standard phytopathological procedure, two representative isolates (SH161 and SH181) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelium, radiate, and the reverse was white. Black pycnidia developed on the sterilized alfalfa stems at 25°C with a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, straight to sinuous, 9.4 to 26.0 × 1.0 to 2.5 μm (n=30). Conidiogenous cells were 2.1 to 15.1 × 0.9 to 2.5 μm (n=30). Alpha conidia were 7.4 ± 0.6 × 2.0 ± 0.2 μm (n=50), hyaline, ellipsoidal to lanceolate. Beta conidia were 29.5 ± 1.8 × 1.1 ± 0.1 μm (n=30), aseptate, hyaline, smooth, curved to hooked. Morphological features of two isolates matched those of spp.. DNA of two isolates was extracted and the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b and CYLH3F/H3-1b. The sequences were deposited into GenBank (Accession Nos. for isolate SH161: OK326730 for ITS, OK413403 to OK413406 for TUB, CAL, HIS and TEF1-α; and isolate SH181: OK331347 for ITS, OK413407 to OK413410 for TUB, CAL, HIS, and TEF1-α). BLAST search of SH161 showed high similarities with sequences of (AR5193) [KJ210529 (ITS), Identities = 438/512, (94%); KJ420850 (HIS), Identities = 466/472, (99%); KJ210550 (TEF1-α), Identities = 345/350, (99%); KJ434999 (CAL), Identities = 344/345, (99%); KJ420799 (TUB), Identities = 508/517, (98%)]. BLAST results of SH181 are 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 SH161 and SH181 in the clade of . Based on the multi-locus phylogeny and morphology, two isolates were identified as . The pathogenicity was tested on 1-yr-old cuttings of var. in the greenhouse. Healthy leaves were wounded with a sterile needle, then inoculated with 5-mm plugs from the edge of two isolates cultures. 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 bidaily to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. Three days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. was reisolated from inoculated leaves. No fungus was isolated from controls. was reported from in Austria. Also, it was reported from and in Ukraine. This is the first report of causing var. leaf spots in China. This finding will provide an effective basis for developing control strategies for the disease.
Yu Wan, Si Wu, Yuan-Zhi Si, De-Wei Li, Li-Hua Zhu

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