First Report of Diaporthe neoviticola Associated with Wood Cankers of Grapevine in Turkey.

Abstract

In recent years, delayed bud bursting, cane bleaching, shoot dieback, and cankers in 1-year-old canes and perennial arms were observed in vineyards of the Aegean region (western Turkey). These symptoms were frequently observed on the following major table grape (Vitis vinifera) cultivars: 'Alphonse Lavallée,' 'Cardinal,' 'Sultana Seedless,' and 'Trakya Ilkeren' in 2012. To determine the causal agents, symptomatic woody tissues (0.5 cm2) were sampled from the canes of nine Manisa and four Salihli Cities (13 total) grapevine varieties and were plated onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). A considerable amount of phomopsis-like fungi were isolated from the symptomatic tissues and fungal colonies were incubated for 2 to 3 weeks to induce sporulation. After incubation for 14 days at 24°C in the dark, white mycelial growth with undulating colony margins, and abundant pycnidia producing hyaline, ellipsoidal, fusoid α-conidia with invisible nuclei, and β-conidia, were observed on PDA, and they resembled species in the Diaporthaceae (1,2). The α-conidia dimensions were 9.3 to 10.2 × 1.9 to 2.9 μm (avg. 9.7 × 2.4 μm) and β-conidia were 19 to 24 × 0.5 to 1 μm (avg. 22 × 0.9 μm). For molecular identification, fungal DNA was extracted from mycelial mats and ribosomal DNA fragments (ITS1, 5.8S ITS2 rDNA, amplified with ITS4 and ITS5 primers) (3) were sequenced and the sequences were compared with those deposited in NCBI GenBank in a BLASTn search. The representative isolate (MBAi43AG) showed 99% homology with Diaporthe neoviticola isolate from New Zealand KC145831.1. The DNA sequence of the identified isolate was submitted to GenBank under accession number KF460427. Pathogenicity tests were conducted under controlled conditions (24°C, 16/8 h day/night, and 70% RH) on 1-cm-diameter, detached green grapevine cv. Cabernet Sauvignon canes (with leaves) using the isolate of D. neoviticola specified above. The shoots were wounded by creating a 5-mm-diameter incision with a sterile scalpel. An agar disc with mycelia and pycniospores was placed into each wound and covered with Parafilm. Sterile PDA plugs were used as mock inoculum for the control plants. There were 10 replicates per treatment and the experiment was repeated twice. After 1 month of incubation, the green shoots were examined for the extent of superficial blackish lesions. The average lesion length on inoculated shoots was 18.2 mm for D. neoviticola. No lesions were observed in the control shoots. The fungal isolate was successfully re-isolated from 96% of inoculated shoots to fulfill Koch's postulates. To our knowledge, this is the first report of D. neoviticola causing wood canker and dieback of shoots on grapevine in Turkey.