LAUSR

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu'er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.