First report of anthracnose on Acer fabric caused by Colletotrichum siamense in China.

Acer fabri Hance, an evergreen tree, is widely cultivated in China for its ornamental value (Lin. 2020). In July 2020, a leaf spot disease, with an incidence of Approximately 48% (12 out of 25), was observed on A. fabri plants (almost 9-year-old) at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E). On average, 30% of the leaves per individual tree were affected. Small spots initially formed along the edge or tip of the leaves and gradually expanded into dark brown spots, and eventually the diseased leaves withered. 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 (Wan et al. 2020). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, LFY-1, LFY-5, and LFY-8 were used for morphological studies and phylogenetic analyses. Colonies on PDA of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 12.8-17.4 ×4.3-5.7 µm (14.3 ± 1.1 × 4.6 ± 0.4 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.3 × 4.7-6.6 µm (7.4 ± 0.3 × 5.5 ± 0.4 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-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. All sequences were deposited into GenBank (ITS, OL818322- OL818324; ACT, OL830175 - OL830177; GAPDH, OL830166 - OL830168; TUB2, OL830163 - OL830165; CHS-1, OL830169 - OL830171; CAL, OL830172 - OL830174). 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 LFY-1, LFY-5, and LFY-8 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 A. fabri plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and inoculated with 10 µL of spore suspension (106 conidia/mL). The spore suspension of each isolate was inoculated onto five leaves. Another three plants inoculated with ddH2O served as the control (Si et al. 2019). All the inoculated leaves were covered with plastic bags to keep a high-humidity for 2 days. 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. The pathogen was previously reported to cause anthracnose on Kadsura coccinea (Jiang et al. 2022), Carica papaya (Zhang et al. 2021), Michelia alba (Qin et al. 2021). This study is the first to report C. siamense causing anthracnose on A. fabric. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.