Identification and Characterization of Eutypa lata on Grapevine in Serbia.

In the period from late May 2004 to late May 2010, grapevine (Vitis vinifera L.) between 11 and 22 years old was observed for the incidence of symptoms of shortened shoots and zigzag internodes, with tiny, chlorotic leaves, torn and bended edges, with necrosis along the edges of leaves and dead internervous tissue. These symptoms are conspicuous especially when the vine is in the F phases of development. Later, in the course of vegetation, the dying of infected shoots and branches or covering of symptoms by a new foliage mass is perceived. Foliar symptoms are initially confined to one arm of infected vines; however, as the disease progresses, symptoms may spread throughout the entire vine. If a cross-section is made of an infected trunk, the canker appears as a wedge-shaped area of discolored wood spreading to the center of the trunk. In the period from 2004 to 2010, disease incidence was high, approaching 20%, and decline-affected cordons, vine branches, or whole plants was very high, resulting in losses of more than 35%. The loss created by a decline in grapevine quality is not included in this estimate. In this study, the causal agent was diagnosed as Eutypa lata (anamorph Libertella blepharis), on the basis of morphology of conidia of a Libertella anamorph on a 4- to 6-week-old culture on PDA (1) and by molecular identification. Molecular identification was performed by PCR and RFLP analysis and supplemented by sequence analysis. Total DNA was isolated from cultured mycelia of fungi using CTAB extraction protocol. PCR reaction was performed by universal ITS1/ITS4; the primer pair and RFLP patterns were determined after restriction with AluI (3). For specific identification of E. lata, the primer pair Lata 1/Lata 2.2 (2) were used and the 385-bp fragment was detected from analyzed isolates. Five selected isolates were purified and a fragment encompassing ITS1, ITS2, and 5.8S rDNA gene was sequenced. Sequences were deposited in the NCBI database under Accession Nos. JQ041699, JQ041700, JQ041701, JQ041702, and JQ041703. Sequence comparisons revealed high nucleotide identity among isolates (99.6 to 100%). When aligned with other E. lata isolates retrieved from the NCBI database, Serbian isolates show the highest nucleotide identity with the isolates from North America (AY462541, AY462540, AY662393, AY662392) and Australia (EU835166, EU835163, EU835162, EU835161, EU835160, EU835159, EU835156). A pathogenicity test was performed in February 2006 in a greenhouse at room temperature (approximately 22°C) and included inoculation rooted cuttings of grapevine (cultivars Cabernet Sauvignon, Prokupac, and Drenak) plants by mycelium. Agar plugs containing mycelium were inserted into 5 mm diameter holes drilled in the main stem of the rootlings and sealed by wrapping with Parafilm. Uninoculated control vines treated with a sterile agar plug were included in the experiment. Foliar symptoms and discoloring of wood beneath and above the inoculation site, inoculated plants, was observed. Reisolation and reinoculation were performed 27 months later, and 54 months later the pathogenicity test was confirmed (4). To our knowledge, this is the first report of death of infected cordons of grapevine by E. lata in Serbia. References: (1) D. A. Glawe et al. Mycotaxon 2:123, 1982. (2) P. Lecomte et al. Appl. Environ. Microbiol. 66:4475, 2000. (3) P. E. Rolshausen et al. Plant Dis. 88:925, 2004. (4) M. Sosnowski et al. Aust. N.Z. Grapegrower Winemaker 493:14, 2005.