RESUMO
Spotted laurel (Aucuba japonica) is a popular ornamental bush (it has two-colored leaves and red berries) and is used outdoors and indoors for decoration in South Korea. Anthracnose reduces the aesthetic value of spotted laurel leaves. In August 2022, anthracnose symptoms were observed on leaves in a park at Jeju Island, South Korea. Approximately 55% of bushes were infected by this disease. Symptoms consisted of round or irregular lesions that initially appeared as black spots and coalesced into larger, black lesions covering whole leaves and twigs. Entire leaves wither and finally die. To identify the putative causal agent, 12 affected leaves were collected, placed in a plastic box containing moist tissue, and incubated at 25 ºC in the dark to obtain conidial mass. Conidial masses were produced on leaf lesions after 2 days, and then 12 morphologically similar fungal isolates were recovered following single the spore isolation technique on solid potato dextrose agar (PDA) (Cai et al. 2009). Ten-day-old colonies were olivaceous gray with immersed perithecia on the upper side and black at the center on the reverse side. Conidia were aseptate, cylindrical with round ends and measured 14.9 - 22.7 × 5.5 - 9.4 µm (n = 80). Appressoria were brown, irregular in shape, and 7.0 - 16.1 × 5.00 - 9.9 µm (n = 50). Asci were eight-spored, banana-shaped, and measuring 60.8 - 123.1 × 13.00 - 18.9 µm (n = 30). Hyaline ascospores were single-celled, curved or straight with round ends, and ranged in size was 15.5 - 23.3 × 5.1 - 11.8 µm (n = 50). The morphological characteristics of the isolates overlapped with those of Colletotrichum species within the C. gloeosporioides complex, including Colletotrichum fructicola (Weir et al. 2012). Five genomic DNA loci of the isolates, including the partial ITS rDNA region, ACT, GAPDH, TUB, and ApMat genes, were amplified and sequenced using ITSF1/ITS4, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting consensus sequences were deposited in the GenBank and the accession numbers (ITS = LC739331- LC739334, TUB = LC739335- LC739338, GAPDH = LC739339- LC739342, ACT = LC739343 -LC739346, ApMat = LC742925 - LC742928) were obtained. A maximum phylogenetic tree was constructed based on the combined data sets of ITS, ACT, GAPDH, TUB, ApMat sequences. The isolates were clustered with reference isolates of C. fructicola (isolates ICMP18581). The pathogenicity test was performed on uninfected, healthy spotted laurel cuttings in the pot. Five leaves per seedling were selected, surface sterilized with 70% ethanol, and rinsed with sterile distilled water (SDW). A sterile pin was used to make 3 to 4 wounds on each side of the leaf from the midrib. 10 µl of spore suspension per wound spot (1 × 106 spores/ml) was applied on the wounds of one site from midrib, and SDW was placed on the wounds of other site as a control. The treated seedlings were covered with sterile plastic bag and kept in a 12-h fluorescent light/dark cycle under greenhouse conditions at 25 ± 2°C and 80% relative humidity. Two seedlings were inoculated with a single isolate, and this experiment was repeated twice. Circular or irregular lesions appeared after 5 days of inoculation, while the control remained asymptotic. Koch's postulates were fulfilled by reisolating and reidentifying the causal agent from the lesions of inoculated leaves. Colletotrichum fructicola has been reported as the causal agent of anthracnose on mango (Joa et al. 2016), apple (Kim et al. 2018), grapes (Lim et al. 2019), peaches (Lee et al. 2020), and hybrid pear (Choi et al. 2021) in South Korea. To the best of our knowledge, it is the first report of C. fructicola causing anthracnose on spotted laurel. This study will be helpful to develop effective management strategies to minimize leaf lesions.
