First report of large-berry coffee () anthracnose caused by in China.

Large-berry coffee (Coffea liberica) is one of the three cultivated coffee species and a precious breeding germplasm in China (Yan et al, 2019). Anthracnose is a damaging epidemic disease on coffee worldwide (Mohammed et al. 2015). Between June and September 2022, anthracnose was observed on coffee plants in Puer area, Yunnan, China and disease incidence (% plants diseased) of 8.5%-28.2% was recorded in the field. The disease symptoms were observed at all growth stages. Lesions on leaves were circular or oval, with a white to gray central zone outlined by a brown margin and surrounded by a chlorotic halo, Φ5.1-18.5 mm; some lesions extended and coalesced later to form large, blighted areas, leading to complete leaf senescence, defoliation and bare blighted branches on heavily infected trees. The spots on coffee berries were oval or fusiform, sunken and brown-black; diseased berries became gray-black and dried-out but remained on the tree. Leaves with typical anthracnose lesions were collected from fields in Simao ( 22.07°E,100.98°N) to isolate the pathogen. Leaf pieces (5×5mm) from the lesion margin were cut, surface-sterilized with 75% ethanol and 2% NaClO, and cultured on PDA at 25°C. Three isolates with the same colony morphology were obtained by hyphal tip purification. Detached and intact leaves of 6-month coffee seedlings were inoculated with Φ5mm mycelial discs of the isolates. Anthracnose lesions developed on the inoculated leaves, with all 3 isolates, 7d after incubation in a growth chamber (25°C, > 90% RH and lighting 8 h/d at 11000 lux). Pathogens with the same colony morphology as those of the original isolates were re-isolated from the infected tissues of inoculated leaves, thus fulfilling Koch's Postulates. The ITS sequence (PP550861) for the isolate was PCR-amplified and Blast-n analyses showed 100 % (554/554bp) identity to Colletotrichum kahawae LWTJ01; so they were the same population and coded as KFTJ02. The actin (ACT), calmodulin(CAL), glyceraldehydes-3-phosphate dehydrogenase (GAPHD) and histone 3 (HIS3) genes (Qiu et al. 2020) were amplified from one of KFTJ02 isolates, sequenced and deposited in NCBI GenBank (OR842543, OR842544, OR842545 & OR842546). A phylogenetic tree was generated based on the concatenated sequences of the four genes and those of related Colletotrichum spp. using MEGA 6.0 and KFTJ02 clustered in the same clade with C. kahawae IMI319418 on the tree (Bootstrap sup.=88%). When cultured at 25°C on PDA for 7 days, its colonies were near round or ovoid, gray-white, contoured, Φ73.2-80.1 (76.2±2.3)mm or growth rate 10.2-11.1(8.1) mm/d (n=10). The hyphae were hyaline, septated, branching at near right angles. Conidial masses formed 14 days after incubation. The conidia were elliptical, hyaline, monocellular, 10.2-15.5 (12.7±1.06)×3.8-5.2 (4.3±0.52) µm (n=50). The appressoria were black-brown, oval or irregular, 7.8-9.3 (8.5±0.81)μm (n= 50). These morphological characteristics were consistent with those of C. kahawae (Bridge et al, 2008). Therefore, KFTJ02 was identified as C. kahawae, which has been found to infect Camellia oleifera, Areca catechu and Ficus microcarpa (Wei et al, 2023; Zhang et al, 2020; Lin 2023). The coffee berry disease pathogen (C. kahawae) is a quarantine species which has not been recorded and so it is first reported on coffee crops in China. Results of the present study provide important references for further studies on this disease.