First Report of Botryosphaeria dothidea Causing Canker on Cotinus coggygria in China

The smoke-tree (Cotinus coggygria) is one of the most important ornamental tree species used in ecological and landscape plantings in China. In summer of 2018, cankers and dieback were observed on C. coggygria grown in several forest parks located in Shandong Province, east-central China. More than 50 trees were observed with the disease. Cankers were located on the trunk, and as disease developed, trees showed a progressive dieback. Tissues below the infected bark revealed dark-brown lesions. To identify the causal pathogen, the phloem and xylem sectors of 10 diseased C. coggygria trees were collected from three locations in Shandong. Pieces of tissue from the border between necrotic and healthy tissues were surface disinfected by immersing in 75% ethanol for 30 s and rinsing three times with sterilized double distilled water, and then the samples were cultured on potato dextrose agar (PDA) and incubated at 25°C for 5 to 7 days. More than 30 isolates were obtained. Developing fungal colonies produced copious, white, aerial mycelium that became dark green with age. Pycnidia developed after about 20 days. Conidia were hyaline, aseptate, fusiform to ellipsoid, and measured a mean dimension of 22 ± 7 × 6 ± 2 μm (n = 20). Fungal identity was confirmed based on comparisons of DNA sequences of the rDNA internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (tef1) and β-tubulin (tub), which were amplified using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone et al. 1999), and BT-2a/BT-2b (Glass et al. 1995), respectively. Six isolates selected for sequence analysis showed 100% identity with each other; hence, only one isolate was deposited in GenBank (ITS, MK168571; tef1, MK722179; and tub, MK720626) and used to perform Koch’s postulates. All sequences blasted against the GenBank database showed 98 to 100% identity to records of B. dothidea (accession nos. MF409167, KU306118.1, and MG878299.1). Fifteen 1-year-old uninfected C. coggygria seedlings used in the pathogenicity experiment were wounded using sterile blades. Mycelial plugs (3 to 4 mm in diameter) of B. dothidea from actively growing colonies were incubated to same-size bark wounds at the central region of stems and wrapped with Parafilm. Sterile PDA plugs were used for control seedlings. Inoculated and control seedlings were inoculated in greenhouse conditions of 15 h light/9 h dark at 25°C with 60% relative humidity and watered as needed. After 2 weeks, all C. coggygria seedlings developed canker lesions on inoculated stems, and no symptoms manifested on the noninoculated controls. The average lesion size caused by fungal inoculations in stems was 9.5 ± 2 cm (n = 10). B. dothidea was recovered from each lesion but not from negative controls, confirming completion of Koch’s postulates. B. dothidea has been reported to cause canker on Aucuba japonica (Zheng et al. 2019) and Carya cathayensis (Zhang et al. 2011) in China. Although B. dothidea has been reported from herbarium specimens as a colonist of C. coggygria in east-central China (Li et al. 2007), to our knowledge this the first report of B. dothidea causing stem canker on C. coggygria in China.