First Report of Onion yellow dwarf virus , Leek yellow stripe virus , and Garlic common latent virus on Garlic in Croatia

Vegetative propagation of garlic often leads to mixed virus infections that cause significant yield and quality reduction. Onion yellow dwarf virus(OYDV), Leek yellow stripe virus(LYSV), and Garlic common latent virus(GCLV) are the most common viruses in Allium species in the Mediterranean region. They are all transmitted by aphids in a nonpersistant manner. Among these three viruses only OYDV has been previously known to occur in Croatia (Stefanac 1977); however, that report concerned onion infections. At the beginning of 2015, symptoms of yellowing and reduced growth were observed on autochthonous garlic cultivars in commercial fields in Zadar County (northern Dalmatia) and in an experimental field in Zagreb (planting material originating from eastern part of Croatia). A total of 213 symptomatic samples (95 from Zadar County and 118 from Zagreb) were collected and analyzed. Plants were initially tested with ELISA for the presence of OYDV, LYSV, and GCLV using commercial kits (Bioreba AG, Switzerland) following manufacturer's instructions and using leaf tissue as a potential source of antigen. The incidence of viruses varied greatly between the two locations. In samples collected from Zadar County, OYDV infections were significant higher (97.9%) than those of GCLV (11.6%) and LYSV (7.4%). On the other hand, all plants from Zagreb were uniformly infected with a mix of the three targeted viruses. One ELISA-positive plant for each virus was selected for further confirmatory tests. To that purpose, we performed the extraction of total nucleic acid (TNA) using QIAGEN Rneasy Plant mini kit (Valencia, Cam USA) according to manufacturer's instructions. After extraction of TNA, presence of viruses was confirmed by one-step RT-PCR using conditions and primers described by Fidan and Baloglu (2009) for GCLV and Dovas et al. (2001) for OYDV and LYSV, resulting in amplicons of 481, 283, and 304 bp, respectively. PCR products, named GCLV-G-Cro, LYSV-G-Cro, and OYDV-G-Cro, were directly sequenced in both directions (GenBank Accession Nos. KT336495, KT336496, and KT336497, respectively). BLAST searches indicated that Croatian isolates were closest to Japanese isolates of GCLV (AB004804) and OYDV (AB000837), and an Argentinian isolate SW9 of LYSV (KF597284) showing 92%, 96%, and 98% nucleotide identity, respectively. Considering the recent expansion of autochthonous garlic cultivars production, the economic importance of these pathogens is likely to increase. To our knowle