Pomegranate, Punica granatum Linn., is widely planted as an ornamental and a fruit crop in Huaiyuan County, Anhui Province, which is the primary pomegranate production area in China. In the early summer of 2012, twig dieback and fruit rot were observed on about 10% and 30% of the pomegranate trees, respectively, in several villages of Huaiyuan County. Necrosis was observed in the twigs, resulting in death of the branches. On fruit, dry rot started at the sepals, covered the entire surface in severely infected fruit, and eventually resulted in shriveling of the fruit. Abundant, black, and solitary pycnidia were observed on diseased twigs and fruit. Pieces of tissue (3 mm in size) from diseased twigs and sepals were surface-disinfected in 75% ethanol for 1 min, washed in sterile water three times, plated on potato dextrose agar (PDA) acidified with 2.5 ml 85% lactic acid per liter, and incubated at 25°C. Resulting fungal cultures produced pale green or white aerial mycelia and sporulated after 5 to 7 days. Pycnidia, ~80 to 130 μm in diameter, were globose and black with thin and membranous walls and contained hyaline, one-celled, and ellipsoid to fusiform conidia, averaging 10.8 to 17.2 × 2.9 to 4.7 μm in size. These morphological features were consistent with Pilidiella granati Sacc. (= Coniella granati Sacc.) (2). Genomic DNA from each of the 10 isolates was extracted and purified using a DNA Gel Extraction Kit (AxyPrep, Hangzhou, China), and PCR was conducted using a DNA Engine System PTC-200 (BIO-RAD, Watertown, MA) with ITS1 and ITS4 internal transcribed spacer universal primers. A single 616-bp fragment was amplified from all 10 isolates and sequenced. Sequence analysis revealed that the ITS from these isolates were identical (100% similarity, GeneBank Accession No. KF560320) to each other and showed >99.5% similarity with those of the P. granti isolates deposited in GenBank (AY339342.1). To evaluate pathogenicity, mycelial plugs (5 mm diameter) from 7-day-old PDA cultures were transferred onto the non-wounded sepals of pomegranate fruit (one plug per fruit, six fruits per isolate), and then all inoculated fruit were placed in plastic bags and maintained at 25°C for 14 days. In addition, twigs on pomegranate plant growing in the field were inoculated by placing mycelial plugs of the fungus on young bark and covered with cotton saturated with sterile water (one plug per twig, six twigs per isolate). Sterile PDA plugs were used as controls in both tests. All 10 isolates colonized the fruit after 5 to 8 days; this was followed by the appearance of dry rot and formation of abundant pycnidia after 10 to 12 days. No decay was observed on the control fruit. Isolates were also pathogenic on twigs, resulting in brown lesions after 2 months that were 2 to 5 cm long. No lesions were observed on the control twigs. Furthermore, the pathogen was isolated from all inoculated fruit and twig tissues and identified to be P. granati as described above, fulfilling Koch's postulates. This pathogen has been reported in Spain (3), Greece (4), and Iran (1), causing crown rot on pomegranate in addition to infecting fruit, but has not been reported previously in Anhui Province of China. This disease is an emerging problem in Anhui Province and will necessitate the development of new disease management practices to sustain commercial production in this region.
References: (1) M. Mirabolfathy et al. Plant Dis. 96:461, 2012. (2) Niekerk et al. Mycol. Res. 108:283, 2004. (3) L. Palou et al. New Dis. Rep. 22:21, 2010. (4) T. Thomidis et al. Plant Dis. 95:79, 2011.
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