LAUSR

Aucuba japonica is a plant with colorful leaves that is widely used in landscaping. Due to its shade tolerance and cold resistance (Li et al. 2013), A. japonica has become a dominant plant species in Guiyang city, China. From 2018 to 2021, an anthracnose disease was observed on A. japonica in Huaxi District (26°23'03'' N, 106°37'58'' E), Guiyang, Guizhou Province. The incidence of anthracnose in newly planted A. japonica reached 90%, resulting in a mortality rate of 30%. This has become a major disease for A. japonica in Guiyang. Typical symptoms include faded spots that initially appear at the infection center, followed by black-brown lesions with irregular edges. At the latter stage, slightly raised black spots are seen arranged in a wheel pattern. In severe cases, diseased leaves fall off. To identify the pathogen, leaf pieces (5 mm × 3 mm) containing symptomatic tissue with healthy margins were excised. The pieces were sterilized with 75% ethanol for 1 min, rinsed three times with sterile water, and cultured on potato dextrose agar (PDA) in Petri dishes at 26°C. White to cream colonies were developed after growth on PDA for 2 days. Mycelial growth ranged from 4.8 to 5.0 mm/day. Conidia were cylindrical, obtuse apex, and protruding base, with dimensions of 18.5 to 20.0 × 7.0 to 9.0 μm (n = 20). Conidial length:breadth ratio was 2-3 (n = 20). Acervuli and sclerotia had not been seen on PDA. To confirm the species of the isolate, PCR was performed on extracted DNA to amplify the ribosomal DNA internal transcribed spacer (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the calmodulin (CAL) gene, and the large subunit (LSU) region by using the primer pairs ITS1/ITS4 (Schoch et al. 2012), GDF1/GDR1 (Guerber et al. 2003), CAL228F/CAL737R (Carbone & Kohn 1999), and NL1/NL4 (Ziemiecki et al. 1990), respectively. The obtained 553 bp ITS sequence (Genbank accession no. MZ424503) showed 99.82% similarity with several C. boninense sequences (MN429163, MN542218, KM520024, and KM520014). The obtained 233 bp GAPDH sequence (OM643394) showed 99.07% similarity with C. boninense (MT602277, GQ221769, MK544890, and MK544888). The obtained 763 bp CAL sequence (OM313386) showed 99.32% similarity with C. boninense (MK569127). The obtained 584 bp LSU sequence (OK513272) showed 99.83% similarity with C. boninense (MH876452, MH877053, and MH876450). Phylogenic trees based on the sequences of ITS, GAPDH, and CAL (Damm et al. 2012), the species of isolate was confirmed as C. boninense. To confirm pathogenicity and to fulfill Koch's postulates, leaves of five A. japonica plants were wounded with a sterilized hypodermic needle and inoculated with 1 μL of a conidial suspension (106 conidia/mL). As control, 5 plants were inoculated with 1 μL of sterile water. After 7 days in a moist climate chamber at 26°C under a 16 h/8 h light/dark cycle, symptoms appeared on all inoculated leaves, while the control leaves remained healthy. The pathogen was reisolated from the inoculated leaves, and was confirmed to be C. boninense using the same morphological and molecular methods as before. C. boninense has been reported to infect many plant species, including Capsicum annuum, Rosa chinensis, and Eucalyptus robusta (Ding et al. 2021; Tozze et al. 2009; Zhang et al. 2018). Another Colletotrichum pathogen, C. fructicola, was reported to cause anthracnose on A. japonica in China (Gong et al. 2016). To our knowledge, this is the first report of A. japonica anthracnose disease caused by C. boninense in Guizhou province, China. Identifying this pathogen provides a foundation to prevent this complex disease and to reduce economic loss.