First Report of Pestalotiopsis camelliae causing Grey Blight Disease on Camellia sinensis in China

Tea gray blight, as one of the most destructive foliar diseases on tea plant (Camellia sinensis [L.] O. Kuntze), has resulted in 17% production loss during peak season in southern India and 10 to 20% yield loss in Japan (Joshi et al. 2009). Tea gray blight disease appears to be caused by a group of Pestalotiopsis-like species that has gone largely unrecognized. In 2015, gray blight disease was observed in several tea plantations located in Chongqing, China. Disease symptoms on tea leaves initially developed as small brown spots that gradually developed to larger, circular to irregular, brown or black lesions. Lesions were covered with black acervuli that changed to a light gray at the late stage of the disease. Twenty isolates collected from symptomatic tea leaves were surface sterilized with 0.1% HgCl2 for 30 s and 75% ethanol for 30 s, and placed on potato dextrose agar (PDA). The colonies on PDA grew fast at 25 ± 2°C and were white to creamy with smooth edge and thin aerial mycelium. The acervuli formed after incubation for 7 to 10 days and were black and gregarious. Conidia were fusiform, straight or slightly curved, and 4-septate. Basal appendages arising from the apex were absent. Numbers of hyaline apical appendages ranged from 3 to 7. The conidia were 19.2 to 33.0 μm long × 3.3 to 6.8 μm wide. The three median cells of all the isolates were concolorous, light to dark brown, 12.5 to 22.3 μm long. Lengths of apical appendages of the isolates were 13.5 to 32.4 μm. All these isolates shared similar morphological characteristics, which were consistent with the description of Pestalotiopsis camelliae associated with gray blight of C. japonica (Zhang et al. 2012). Three isolates were used for DNA extraction. The genomic DNA obtained from cultures grown on PDA for 5 days was amplified with the primers ITS4/ITS5 for the partial ITS region, T1/βt2b for the partial β-tubulin gene region (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997), and EF1-526F/EF1-1567R for the partial TEF gene region (Maharachchikumbura et al. 2014). The gene sequences (KY319135, KY363539, and KY342363) of ITS, β-tubulin, and TEF regions of three representative isolates were identical and shared 100, 99, and 99% sequence homology with the isolate of P. camelliae CBS 443.62 (KM199336 and KM199424) from C. sinensis in Turkey and the isolate of P. camelliae OP111 (JX399074) from C. japonica in China, respectively (Maharachchikumbura et al. 2014; Zhang et al. 2012). Pathogenicity tests were performed on five detached tea twigs (∼20 leaves) with a spore suspension (1 × 108 conidia/ml) prepared using isolates grown on PDA for 10 days at 25 ± 2°C. Before inoculation, leaves were surface disinfected with 75% ethanol and wounded with a sterile needle. Controls were inoculated with sterilized distilled water over the wounds. All inoculated tea twigs were placed in glass culture dishes and maintained in a growth chamber with 12 h light cycles and 100% RH at 25 ± 2°C. Experiments were repeated three times. Small brown lesions were observed surrounding the inoculation sites of wounded leaves after 4 days, with symptoms continuing to develop, which resembled field symptoms. Symptoms were never observed on controls. The fungi reisolated from the lesions of diseased tea leaves were identical to the original isolates. This is, to our knowledge, the first description of P. camelliae causing gray blight disease on C. sinensis in China based on the morphological traits and multigene data of ITS, β-tubulin, and TEF. © 2017, American Phytopathological Society. All rights reserved.