First Report of Botryosphaeria dothidea causing leaf necrosis of Camellia sinensis in Fujian Province, China

Camellia sinensis (L.) Kuntze (Theaceae), commonly known as tea, is a major economic crop cultivated in China (Maharachchikumbura et al. 2013). A cultivar of C. sinensis, purple rose, is commonly grown in the tea base of Zhangzhou, Fujian Province. In June 2015, a leaf necrosis was observed on this cultivar in different farms of different counties in Fujian Province. Symptoms began as small brown lesions on young and mature leaves, becoming necrotic, 2 to 4 cm diameter with a brown margin. To determine the causal agent, symptomatic leaf tissue was collected from plants from Zhangzhou in June 2015. Small pieces from the margin of the necrosis were surface sterilized in 75% alcohol for 2 min, 5% NaClO for 1 min, rinsed twice in distilled water, plated onto potato dextrose agar (PDA), and incubated at 25°C for 5 days. Among the isolated pathogens (such as Colletotrichum) from the leaves, several strains (8) were identified as Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not. from morphological characters. The cultures produced white-gray fluffy mycelia, black from below, becoming dark brown with age. Conidiomata were produced after 10 days on PDA. Conidiogenous cells were hyaline, subcylindrical, and 4 to 16 × 2 to 5 µm (n = 20). Conidia were 14.6 to 26.1 × 4.8 to 8.8 µm (n = 40), hyaline, unicellular, narrowly fusiform, with a subtruncate to bluntly rounded base, forming a septum before germination, and smooth walled with a granular content. DNA was amplified using rDNA-ITS primer pair ITS1/ITS4 (White et al. 1990) and tef1 728F/986R (Carbone and Kohn 1999; Hyde et al. 2014). Maximum parsimony analysis (MP) using PAUP (phylogenetic analysis using parsimony) v. 4.0b10 and a Bayesian inference (BI) was used to construct the phylogenies using Mr. Bayes v. 3.1.2 to confirm the identity of the isolates. Two representative isolates JZB310001 and JZB310002 clustered together with B. dothidea (CBS 115476) with 90% bootstrap support and 0.98 Bayesian posterior probabilities (PP) support. Sequences of the two isolates have been deposited in GenBank under KT459354, KT459355, KT459356, and KT459357, respectively. Based on the above morphological and molecular data, the two isolates were identified as B. dothidea. In order to confirm the pathogenicity, Koch’s postulates were conducted using two methods. In the first method, 5 ml of a conidial suspension (1 × 106 conidia/ml) was sprayed onto 10 detached leaves; five with wounds and five unwounded. Control leaves (five) were sprayed with distilled water. The second method used mycelium plugs (Yan et al. 2013) of actively growing colony of B. dothidea on 10 detached twigs, five with wound and five unwounded. The control twigs (five) were treated with sterile PDA mycelium plug. The inoculated leaves and twigs were maintained in a greenhouse at 28°C at a constant relative humidity of 95%. Brown necrotic lesions appeared on leaves and twigs after 7 days, while the control plants remained healthy. The experiments were conducted three times and the fungus was reisolated was identified as B. dothidea by morphology and molecular data. This is the first report of B. dothidea causing tea leaf necrosis in China. Tea growers should pay more attention in the incidence of this disease. © 2016, American Phytopathological Society. All rights reserved.