First Report of Botryosphaeria dothidea Causing Leaf Spot and Wilt on Celtis sinensis in China

Celtis sinensis Pers. (Chinese hackberry), belonging to the family Ulmaceae, is widely used as a street tree or landscape plant because of its longevity and aesthetic growth habit. Additionally, C. sinensis is of economic importance due to its medicinal properties. Roots and bark of the plant can be used in natural medicine for the treatment of lumbago, measles, tumor, etc (Zhang et al. 2016). In July 2019, symptoms of leaf spot were observed on C. sinensis in Yuanshan national forest park of Zibo, Shandong Province, China (36.48°N, 117.84°E). We surveyed more than 500 square meters of forest area, and more than 80% of the acreage was affected with the leafspot disease. Symptoms on infected leaves appeared as regular round or oval spots, colored in yellow with brown borders, which coalesced into larger spots as the disease progressed. To investigate the cause, 20 leaves of infected tissues were cut into ~2 mm pieces and surface disinfected with 75% ethanol for 30 s, rinsed three times with sterile deionized water. These were air dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 to 7 days. A minimum of 15 isolates were obtained and cultures were initially white, gradually becoming gray green to dark after 1 week, producing copious amounts of gray aerial mycelium. Three representative single isolates were used for molecular identification, which were verified based on the amplification of DNA sequences of internal transcribed spacer region, translation elongation factor 1 alpha and beta-tubulin genes, using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and BT-2a/BT-2b (Glass and Donaldson 1995), respectively. The sequenced genes (GenBank accession no. MT367874, MT385087, MT374083) exhibited 99.63% (Identity=545/547), 99.00% (Identity=297/300), and 100.00% (Identity=451/451) homology with the corresponding genes of type specimen of Botryosphaeria dothidea strain CBS110302 (GenBank accession no. AY259092, AY573218, EU673106), respectively. Morphological and molecular results showed that the isolates were B. dothidea (Slippers et al. 2014; Zhai et al. 2014). Pathogenicity was confirmed using five living, healthy C. sinensis plants with three leaves were wound inoculated with mycelial plugs (about 4 mm in diameter) of B. dothidea from a 7-day-old culture grown on PDA, while inoculated with sterile PDA plugs on the same leaves were served as negative controls. All the plants were covered by plastic sheeting and keep high relative humidity by adding water in time. Seven days later, all inoculated leaves appeared as round dark brown spots, which were larger than observed in the field. The pathogenicity test was repeated three times. No symptoms were observed on negative controls. Fungi re-isolated from inoculated leaves were confirmed as B. dothidea on the basis morphology and molecular characterization as described above. To our knowledge, this is the first report on the presence of B. dothidea affecting C. sinensis plants in China. This discovery is important to ensure the sustainable production of C. sinensis, an important landscaping and medicinal tree.