First Report of Corynespora cassiicola Causing Leaf Spot Disease on Buxus sinica subsp. sinica var. parvifolia in China

Buxus sinica subsp. sinica var. parvifolia, a broad-leaved evergreen plant, is a rare landscaping tree species in China. In September 2017, a black spot disease was observed on B. sinica subsp. sinica var. parvifolia plants in a cultivated field in Jinjing Road, Tianjin (39°13′ N; 117°20′ E), China. Symptoms on leaves were initially small round dark spots, expanding to form subcircular to irregular, 6 to 12 mm diameter, black lesions. As the disease progressed, the infected leaves became chlorotic and dropped prematurely. The stems were asymptomatic. Morphological characteristics of the pathogen were observed with a microscope directly on the infected leaves. Mycelia were branched, septate, hyaline, smooth-walled, and 2.3 to 6.8 μm wide (n = 50). Stromata were absent. Conidiophores were simple or occasionally branched, pale to dark brown in color, and 47.2 to 227.9 × 4.2 to 8.7 μm in size (n = 50). Those were smooth, septate, and formed with 0 to 5 successive cylindrical proliferation. Conidiogenous cells were integrated, monotretic, scar thickened, and 4.1 to 4.5 μm wide (n = 50). Conidia were borne singly or in short chains, and were variable in shape, obclavate to cylindrical, straight or curved, subhyaline to brown in color. The average size of 50 conidia were 38.4 to 122.7 × 8.8 to 14.5 μm, with 4 to 11 pseudosepta. The diseased leaf was dissected into small pieces from margins of individual lesions. These pieces were surface-sterilized in 75% ethanol for 45 s, rinsed twice in sterile distilled water, and placed on potato dextrose agar (PDA). Fungal colonies of similar morphology were selected and transferred to PDA. Colonies grown on PDA media at 25 ± 1°C produced gray to dark brown mycelia. The fungus was identified as Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei based on morphological and cultural characteristics (Ellis and Holliday 1971). For pathogenicity testing, 20 healthy leaves of B. sinica subsp. sinica var. parvifolia from four rooted cuttings were inoculated with conidial suspension (1 × 10⁵ conidia per ml) of isolated pathogen brushed from a 7-day-old culture of the fungus. Blank controls were 10 healthy leaves, from two rooted cuttings, sprayed with sterilized water. Each treatment repeated three times. All inoculated leaves were kept in an artificial climate chamber at 25 ± 1°C, 90% humidity. After 7 days, typical leaf spots were observed on all inoculated leaves while the control leaves displayed no symptoms. The pathogen reisolated from artificially inoculated leaves, had identically morphological conidia to C. cassiicola, fulfilling Koch’s postulates. To further identify the fungus, the internal transcribed spacer region was amplified with primers ITS1/ITS4. The obtained sequence of 497 bp was sequenced (GenBank accession no. MJ518227), and showed 100% identity to sequences of known C. cassiicola isolates (KX638445) in GenBank. C. cassiicola is a common plant fungus with a wide host range (Dixon et al. 2009). In recent years, many new diseases on ornamental plants caused by C. cassiicola have been reported (Farr and Rossman 2015). To our knowledge, this is the first report of leaf spot disease caused by C. cassiicola on B. sinica subsp. sinica var. parvifolia and Buxus spp. in China.