First report of Ilyonectria liriodendri associated with black foot disease of grapevine in Turkey

Grapevine trunk diseases have been an increasing problem of Turkey vineyards in recent years. A study was conducted in nine Chardonnay (grafted on 110R) vineyards to determine the incidence and the causal agents of grapevine trunk diseases in Sarkoy, Tekirdag (northwest of Turkey) in 2014. The vines with tiger-striped leaves, and declining, local drying, or dead arm symptoms (27.3% of all inspected 23291 vines) were recorded and branch, trunk, and rootstock samples (three samples from each vineyard) were taken for laboratory examinations. Dark brown and black spots in cross sections and blackish streaks in longitudinal sections were observed in wood of some rootstocks. Symptomatic tissues were surface disinfested with 95% ethanol and flame sterilized. The internal tissues were plated onto potato dextrose agar amended with tetracycline (0.01%). Petri dishes were incubated at 24°C in the dark for 18 days and floccose to felted colonies, which varied in color from dark orange to dark brown with age, developed (in one vineyard, with 24.3% isolation frequency) around wood chips. Pure cultures of the isolate (MBAi232) produced 1 to 3-septate, straight or slightly curved, cylindrical macroconidia (32 to 38 × 4.9 to 5.7 µm) and a few microconidia. Chlamydospores were brown, ovoid (12 to 17 × 10 to 17 µm), and mostly in short and intercalary in chains. According to morphological characteristics, the isolate resembled Ilyonectria liriodendri with species nomenclature based on the work of Halleen et al. 2006. For molecular identification, fungal DNA was extracted from mycelium and ribosomal DNA fragments (ITS1, 5.8S ITS2 rDNA) and ß-tubulin gene were amplified with ITS 4-5, Bt 2a-2b primers (White 1990; Glass and Donaldson 1995) and sequenced. The sequences were compared with those deposited in GenBank and the isolate showed 99% similarity with I. liriodendri isolates JF735261 (ITS) and KF511973 (ß-tubulin). The sequences were deposited as NCBI GenBank Accession Nos. KP271987, KP271986 for ITS, and ß-tubulin, respectively. Pathogenicity tests were conducted under greenhouse conditions (24°C, 16/8h day/night, 70% RH) on own-rooted grapevine (Vitis vinifera) seedlings cv. Chardonnay using the isolate of I. liriodendri. Plants were removed from the rooting bench, and the roots were slightly trimmed and dip-inoculated with a 106/ml conidial suspension of the isolate for 60 min (Cabral et al. 2012). After inoculation, the rooted cuttings were planted in 1-liter bags containing a mixture of soil, peat, and sand (2:1:1, v/v/v), and maintained in the greenhouse. Sterile distilled water was used for inoculation of negative control plants. Ten vines were inoculated with the isolate and 5 vines were not inoculated. The experiment was repeated twice. After 4 months of incubation, seedlings were examined for vascular discoloration and recovery of fungal isolate. Blackish-brown discoloration of xylem vessels and necrosis in the basal ends and roots were visible in inoculated but not in uninoculated control seedlings. Slight leaf wilting was also observed in some plants. The fungal isolate was successfully reisolated (by 70.5%) from inoculated but not from uninoculated ones. To our knowledge, this is the first report of I. liriodendri causing black-foot disease of grapevine in Turkey. © 2015 The American Phytopathological Society