Yunnan Province is the major region for coffee (Coffea arabica) cultivation in China, contributing to over 98% of the national yield and total production value (Ma et al. 2022). In May 2023, brown spot symptoms were observed only on the leaves of coffee plants in a field located in Baoshan City (98°52'37.988400"E, 24°58'17.673600"N), Yunnan Province. Notably, brown and irregularly shaped spots initially started on the leaf bases. The spots enlarged and developed concentric rings with dark brown margins, which are often surrounded by yellow halos. Finally, the necrotic spots spread across the entire leaf and caused the leaf to curl and fall off. The incidence of the disease was approximately 3% of the coffee plants (n = 600). The symptomatic leaves collected from 10 plants were sectioned (5 × 5 mm), subjected to surface sterilization with 70% ethanol for 40 s, rinsed with sterile distilled water, air-dried, and transferred to potato dextrose agar (PDA). Fungi with grayish-white, cotton-like aerial mycelia grew after 7 days at 28°C. The older mycelia of these isolates displayed dark gray pigmentation. Single conidia were cultivated on PDA, and 15 morphologically similar monosporic isolates were ultimately obtained. Microscopic observation revealed that these isolates produced branched, septate, transparent and amber mycelium. Brown, elliptical or pear-shaped conidia with 2 to 4 transversal septa and 0 to 3 longitudinal septa, measuring 9.6 to 33.3 long × 6.0 to 15.0 μm wide (n = 30), were observed on potato carrot agar (PCA). Molecular identification of multiple genes, such as ITS (Schoch et al. 2012), RPB2 (O'Donnell et al. 2010) and GAPDH (Berbee et al. 1999), indicated consistent 100% identity among these isolates. Sequences of the representative isolates CFSY1-CFSY5 were deposited in GenBank (acc. nos.: OR351112, PP188577, PP188578, PP294863, PP294864, OR509742, PP215341-PP215344, OR509740 and PP239378-PP239381), revealing 98.35% - 100% homology with distinct Alternaria alternata strains previously deposited in GenBank (acc. nos.: PP110780, MN649031 and OR485338). The multigene phylogenetic analysis positioned isolates CFSY1-CFSY5 within a distinct cluster, alongside diverse A. alternata isolates. Based on morphological and molecular characterizations, the pathogen was identified as A. alternata. To verify its pathogenicity, a conidial suspension (1×106 conidia/mL) of isolate CFSY1 was sprayed on six leaves of three healthy one-year-old C. arabica seedlings. Subsequently, the inoculated seedlings were covered with plastic bags and placed in a growth chamber under controlled conditions (a 14 h daylight period and a 10 h dark period at 28°C). The experiment was repeated three times. After 20 days, typical brown spot symptoms analogous to those originally observed in the field appeared on the leaves in all inoculated plants. Reisolation, morphology identification and DNA sequencing substantiated Koch's postulates. In contrast, control plants treated with sterilized water remained asymptomatic, and no pathogen was reisolated from them. Significantly, A. alternata has been previously reported as the causal agent for leaf spot disease in a diverse variety of woody plant species in China, including Prunus avium (Ahmad et al. 2020), Magnolia grandiflora (Liu et al. 2019) and citrus (Wang et al. 2010). This study represents the first report of brown leaf spot caused by A. alternata specifically on C. arabica in China, enriching the contents of fungal pathogens under Chinese coffee cultivation conditions.
Keywords: Alternaria alternata; Coffea arabica; leaf spot.