First Report of Anthracnose Caused by <i>Colletotrichum reniforme</i> on <i>Boehmeria nivea</i> in Sichuan Basin, China

Ramie (Boehmeria nivea), native to China, is a natural fiber-yielding crop. The ramie fibers are long, pure white in color, silky in texture, and highly durable and hygroscopic (Angelini and Tavarini 2013). In May 2025, anthracnose symptoms were observed on 10% of cultivated ramie plants in a 10-ha field, Dazhou City (30.86°N, 107.33°E), Sichuan Province, China. Leaf tissues adjacent to and including lesions were excised, superficially disinfected with 70% ethanol for 20 s and 1% NaClO for 40 s, and washed with sterile distilled water at least five times. The disinfected tissues were incubated on PDA amended with streptomycin sulfate (50 mg/L) in the dark at 25 ℃. Two or three days later, hyphal tips from the edges of growing colonies were transferred to fresh PDA plates. Three representative isolates, G21, G22, and G23, showed identical morphological characteristics. The colonies exhibited cottony aerial mycelia on SNA plates. The upper and lower surfaces of mycelia were initially grayish-white and gradually became brownish-white. Setae were observed on the hyphae. Asci were 60.5 ± 5.4 × 13.7 ± 1.5 µm in size (n = 20), eight-spored, fasciculate, and clavate. Ascospores were 24.2 ± 3.6 × 5.1 ± 0.7 µm in size (n = 30) and slightly curved with obtuse to slightly rounded ends. Meanwhile, colonies produced abundant conidia, which were unicellular, hyaline, aseptate, smooth-walled, straight, cylindrical and rounded at both ends, measuring 18.1 ± 1.5 × 5.5 ± 1.2 µm (n = 30). These morphological characteristics match those of Colletotrichum species (Liu et al. 2022; Xue et al. 2020). Three isolates were properly preserved in our lab. All the isolates were further identified by sequencing rDNA internal transcribed spacer (ITS) regions, actin (ACT), beta-tubulin (TUB2), histone3 (HIS), chitin synthase (CHS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, T1/Bt2b, CYLH3F/CYLH3R, CHS-79F/CHS-345R and GDF1/GDR1 (Liu et al. 2022), respectively. BLASTn searches indicated our ITS (PX090878-PX090880), ACT (PX092338-PX092340), TUB2 (PX092341-PX092343), HIS (PX092350-PX092352), CHS (PX092344-PX092346) and GAPDH (PX092347-PX092349) sequences showed 99.48-100% identity to the corresponding sequences of C. reniforme LC8230 (MZ595847.1, MZ664145.1, MZ673968.1, MZ673867.1, MZ799290.1, and MZ664110.1). Based on concatenated ITS, ACT, TUB2, HIS, CHS, and GAPDH sequences, the constructed phylogenetic tree of Colletotrichum species confirmed that our isolates were C. reniforme. In the pathogenicity test, healthy leaves of ramie seedlings were sprayed with conidial suspension (1 × 105 conidia/mL) of G21, with controls treated with sterile dH2O. Each treatment was incubated in a greenhouse (at 25°C under 90% relative humidity and a 12/12 h light/dark cycle). The experiment was repeated three times. Ten days after inoculation, anthracnose symptoms appeared on inoculated leaves, while controls remained healthy. Koch's postulates were fulfilled by re-isolation of C. reniforme from diseased leaves, based on morphology and molecular methods described above. C. gloeosporioides (Wang et al. 2010) and C. higginsianum (Wang et al. 2011) were previously reported as causal agents of anthracnose on ramie. To our knowledge, this is the first report of C. reniforme causing anthracnose of ramie worldwide. Our study will assist in monitoring the diversity of infectious agents causing ramie anthracnose in China.