First Report of Cryptovalsa ampelina and Eutypella leprosa Associated with Grapevine Trunk Diseases in Chile.

Abstract

Trunk diseases (TD) of grapevines (Vitis vinifera L.) have increased considerably in Chile with an incidence of more than 25% found in ≥7-year-old vineyards. Only species of Botryosphaeriaceae, Phaeoacremonium, and Phaeomoniella were associated with TD in Chile (1,2). From 2009 to 2010, isolations were made from the grapevines 'Cabernet Sauvignon', 'Carmenere', 'Flame Seedless', and 'Pinot Noir' collected in central Chile (33°27' to 34°39'S, 71°17' to 71°33'W). These grapevines showed cankers and vascular necrosis of trunks, arms, and spurs along with a general decline and dieback. Isolations were performed in potato dextrose agar (PDA) plus 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (Sigma-Aldrich, St. Louis, MO), for 14 days at 20°C. On the basis of colony morphology and conidia production, two Libertella-like species were obtained in 26 (7.8%) of 335 trunk samples. On the basis of the internal transcribed spacer region (ITS4 and ITS5) of rDNA, Cryptovalsa ampelina (Nitschke) Fuckel (GenBank Accession Nos. HQ694976 and HQ694977), and Eutypella leprosa (Persoon) Berlese (HQ694974 and HQ694975) were identified, showing 98 to 100% similarity with the sequences of C. ampelina (GQ293913) and E. leprosa (AJ302463.1). C. ampelina produced white-to-creamy, smooth colonies with a creamy underside. Colonies of E. leprosa were white-to-gray with a white underside. Orange conidial masses were exuded after 30 days at 20°C. Conidia on PDA (n = 20) were unicellular, hyaline, filiform, slightly curved, and (19.8) 23.4 ± 2.6 (28.3) × (1.1) 1.4 ± 0.2 (1.8) μm and (19.2) 23.9 ± 3.0 (27.6) × (1.0) 1.2 ± 0.1 (1.5) μm for E. leprosa and C. ampelina, respectively. Stromatic perithecia of C. ampelina, embedded in the bark, were observed in dead pruning residues of infected vines (4). Pathogenicity tests were conducted with two isolates of each species, on 30-day-old 'Carmenere', rooted in vitro (n = 12), that were inoculated by placing a 5-mm agar plug on the surface of the propagation medium. Additionally, 15 cm long pieces (n = 5) of 1-year-old canes from 'Carmenere', 'Chardonnay', and 'Red Globe' were inoculated by placing a 5-mm agar plug underneath a cut aseptically made in each cane. An equal number of noninoculated plants and canes, but treated with sterile agar plugs, were used as controls. Leaf number (LN), shoot length (SL), and root length (RL) were assessed on plants in vitro after 28 days at 20°C. The extent of vascular discoloration (VD) obtained in canes was determined after 45 days in humid chambers at 20°C. One-way analysis of variance was performed and mean differences were studied by Tukey's test. C. ampelina and E. leprosa significantly (P < 0.05) reduced the LN, SL, and RL relative to the control plants. They also caused a VD of 10.1, 11.6, and 9.8 mm and 11.2, 13.4, and 10.0 mm in 'Carmenere', 'Chardonnay', and 'Red Globe', respectively. No symptoms were observed on the control canes. C. ampelina (100%) and E. leprosa (75%) were reisolated from inoculated plants and canes. To our knowledge, this is the first report of C. ampelina and E. leprosa on grapevines in Chile. However, their relative importance as causal agent of trunk disease remains to be determined. C. ampelina and E. leprosa have been associated with grapevine cankers in the United States and Spain (3,4).