First Report of Stem Canker on Cherry Caused by Phomopsis perniciosa in Shandong Peninsula, Eastern China.

Cherry is a main fruit tree species in Shandong Peninsula, which is one of the most important cherry-production areas of China. A stem canker disease was first noted in a 15-year-old cherry orchard in Yantai, Shandong Peninsula in May 2009. Canker and branch dieback were the main symptoms of the disease and cracks often appeared at the margins of sunken cankers, which exposed the wooden stem. In later stages from April to May, black pycnidia were observed on the surface of cankered bark and cirri containing α-conidia were extruded under wet conditions. Wooden tissue under the diseased bark was dark brown, in contrast to the healthy tissue that was yellowish green. On the basis of morphological characteristics, the pathogen was putatively identified as Phomopsis perniciosa (1). Pycnidia were smaller in naturally infected branches than when produced on potato dextrose agar (PDA) medium (180 to 365 × 65 to 226 μm). Cultures of the pathogen appeared creamy white with concentric rings on PDA at 25°C and a mass of α-conidia (5.75 to 11.13 × 2.08 to 3.46 μm) and β-conidia (31.24 to 34.68 × 1.45 to 1.82 μm) were produced within 3 weeks. Alpha-conidia were hyaline, fusiform-elliptic to oblong-elliptic, and biguttulate. Beta-conidia were hyaline and unicellular, filiformia, leviter arcuata vel hamata. Total DNA was extracted from three monoconidial isolates collected from different infected trees. The internal transcribed spacer (ITS) region was amplified using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-CCTCCGCTTATTGATATGC-3'). The ITS amplicons were sequenced (582 bp) from three isolates and no nucleotide variation was observed. BLAST analysis of the obtained ITS sequences showed that isolate 230101 had 99% homology with a Phomopsis sp. (GenBank Accession No. AB302248) isolated from fruit trees in Japan. The nucleotide sequence from isolate 230101 has been deposited in GenBank (Accession No. JF812647). Pathogenicity of the isolate was confirmed by inoculating branches of 3-year-old cherry trees with either conidia or hyphae. Inoculations were performed by making an incision with a sterile scalpel at the dissected area to expose the tissue under the bark. An agar plug (4 × 4 mm) containing 5-day-old cultured hyphae or 50 μl of a conidium suspension containing 10 α-conidia per ml was placed on each of the inoculation sites, wrapped with moist cheesecloth, and sealed with Parafilm. Control trees were treated similarly with sterile blocks of PDA or water, respectively. For each inoculation technique, five shoots were inoculated and the inoculation treatments were replicated three times. All inoculated and control trees were kept in a greenhouse and watered as needed. After 10 days, cankers and necrotic lesions developed on all shoots inoculated with P. perniciosa and the control trees did not display any symptoms. The same pathogen was reisolated from symptomatic branches. Phomopsis spp. are known to cause cankers and dieback of several woody hosts (2), but no reports have been found that the pathogen causes cherry canker and dieback in China. References: (1) P. K. Chi et al. Flora Fungorum Sinicorum-Phomopsis 34:127, 2007. (2) D. P. Weingartner and E. J. Klos. Phytopathology 65:105, 1975.