First Report of <i>Colletotrichum truncatum</i> Causing Anthracnose on <i>Coffea arabica</i> in Yunnan, China

Coffee (Coffea arabica L.) is an important economic crop in China and globally. Approximately 98% of China's coffee is grown in Yunnan Province. In February 2024, anthracnose was observed in a coffee nursery in Dehong, Yunnan (24°23′55.73″N, 97°59′22.31″E), with an incidence rate of 5%. Symptoms initially occurred on leaf margins or tips as water-soaked lesions, subsequently expanded into large, sunken, irregular brown blight lesions with black acervuli. Leaf samples exhibiting typical lesions were collected from five plants for pathogen isolation. Leaf pieces (5 × 5 mm) were cut from the lesion margins, surface sterilized with 75% ethanol for 20 s and 2.5% sodium hypochlorite for 30 s, rinsed three times with sterile distilled water and blotted dry with sterile filter paper, then plated on potato dextrose agar (PDA) and incubated at 25 °C for 4 days. Five morphologically similar pure isolates (YDL1 to YDL5) were obtained. Colonies were circular with regular margins, presenting sparse and appressed aerial mycelia, and reached 6.5 cm in diameter after 7 days. Colony surface turned from white to pale gray and produced abundant acervuli. Conidia were hyaline, unicellular, aseptate, and falcate, with distinctly curved and acute apices, granular contents and a prominent transparent central region, measuring 20.5 to 27.4 × 2.8 to 4.6 μm (n=50). Appressoria were light brown, elliptical to clavate, with intact or slightly lacerated edges. Setae were brown, 51.2 to 185.9 μm long, 4.2 to 6.5 μm diam, 2-to 5-septate, and tapering slightly toward acute to rounded apex. These morphological characters were similar to those of Colletotrichum truncatum (Damm et al. 2009). A spore suspension (106 conidia/mL) was prepared from 14-day-old cultures grown in the dark at 25℃. Then, 20 μL of the suspension was dropped onto wounded coffee leaves with a sterile needle. For each isolate, three healthy plants were inoculated, and another three treated with sterile water as controls. All plants were kept separately in an incubator(25 °C, 90% RH, 12-h photoperiod). Seven days post-inoculation, all inoculated leaves developed symptoms consistent with field observations, whereas no symptoms appeared on control leaves. Koch's postulates were fulfilled by reisolating the pathogen from lesions of inoculated plants and confirming morphological consistency with the original isolate. YDL1 and YDL2 were selected for molecular identification. Five genomic regions, including the internal transcribed spacer (ITS), β-tubulin, chitin synthase (CHS), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified with primer pairs ITS1F/ITS4, T1-F/TUB4Rd, CHS-79F/CHS-354R, ACT-512F/ACT-783R, and GDF1/GDR2, respectively. Sequences were deposited in GenBank (ITS: PX317819 for YDL1 and PX317820 for YDL2; ACT: PX363488 and PX363489; CHS: PX363490 and PX363491; GAPDH: PX363492 and PX363493; β-tubulin: PX363494 and PX363495) and showed 99 to 100% similarity with C. truncatum strains. In the phylogenetic tree based on ITS, GAPDH, CHS, ACT, and β-tubulin gene sequences, the isolates YDL1 and YDL2 clustered with C. truncatum CBS 151.35. Based on combined morphological, molecular and pathogenicity analyses, the pathogen was identified as C. truncatum. This pathogen has been reported on melon and tobacco in China (Bi et al. 2017; Shi et al. 2023). To our knowledge, this is the first report of C. truncatum causing anthracnose on C. arabica in Yunnan, China, as well as worldwide.