First report of leaf spot caused by on in China.

Nageia nagi (Thunb.) Kuntze is widely cultivated in China for its ornamental and economic value. In August 2019, a leaf spot was observed on N. nagi plants at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E). Disease incidence was about 35%, and the diseased leaf rate was above 40%. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became reddish-brown, eventually developing large irregular lesions. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C (Zhang et al. 2021). Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-six isolates of Colletotrichum ssp. were obtained (isolation frequency about 82%). Three representative single-spore isolates (ZB-1, ZB-3, and ZB-7) were used for morphological studies and phylogenetic analyses. Colonies on PDA medium of the three isolates were white to gray in color with cottony mycelia. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.1-17.9 ×4.4-6.8 µm (15.6 ± 1.2 × 5.4 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.7-9.3 × 4.6-6.9 µm (7.8 ± 0.2 × 5.6 ± 0.3 µm, n=100). Morphological features were similar to Colletotrichum siamense complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), β-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. Sequences of them deposited in GenBank under nos. OL826760 - OL826762 (ITS), OL830205 - OL830207 (ACT), OL830196 - OL830198 (GAPDH), OL830193 - OL830195 (TUB2), OL830199 - OL830201 (CHS-1), and OL830202 - OL830204 (CAL). A Blast search of GenBank showed that ITS, ACT, GAPDH, TUB2, CHS-1, and CAL sequences of the three isolates were identical to Colletotrichum siamense at a high level (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 ZB-1, ZB-3, and ZB-7 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. The pathogenicity of three isolates was tested on six N. nagi plants (three for inoculation, three for controls), which were grown in the field. Six healthy leaves were wounded with a sterile needle and inoculated with 10 µL of conidial suspension (1 × 106 conidia/mL) per plant. Healthy leaves were inoculated with ddH2O as a control by the same method. All the inoculated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiment was repeated three times. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Up to now, Cephleuros virescens, Pestalotiopsis longisetula, Alternaria tenuissima, A. alternate, and Phoma glomerata could infect N. nagi (Zhou et al. 2015; Zhang et al. 2016), and cause leaf spots in China. To our knowledge, this is the first report of C. siamense causing leaf spots on N. nagi worldwide. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.