First Report of Beet yellows virus (BYV) on Sugar Beet in Croatia

Abstract

Sugar beet (Beta vulgaris var. saccharifera L.) is the main crop used for the production of sugar in Croatia. Beet yellows virus (BYV), type member of the genus Closterovirus, is characterized by flexuous, filamentous particles which are transmitted by several aphid species in a semipersistent manner (Agranovsky and Lesemann 2000). This widely spread virus is reported from most of the sugar beet-growing areas of the world, including Europe. In the geographic region that was once the former Yugoslavia, the presence of BYV was suspected based on field symptoms reported from Serbia (Nikolic 1951) and Croatia (Panjan 1951). During the 2013 and 2014 growing season, the presence of sugar beet plants with symptoms of yellowing was sporadically observed in commercial fields located in eastern Croatia (i.e., Tovarnik, Bosnjaci, and Virovitica). In late July 2014, a significant number of sugar beet plants, located mainly on the field edges, with the aforementioned symptoms were noticed in experimental fields of various sugar beet cultivars located at the University of Zagreb, Faculty of Agriculture. In total, 102 plants collected from commercial fields and 12 plants from the university experimental fields with cvs. Torda and Libero were selected and screened using ELISA for the presence of Beet necrotic yellow vein virus (BNYVV), Beet mosaic virus (BtMV), Beet mild yellowing virus (BMYV), Beet western yellows virus (BWYV), and BYV. To conduct the diagnostic assays, commercial ELISA kits from Loewe Biochemica GmbH (BNYVV, BtMV, and BYV) and Sediag (BMYV and BWYV) were used. BYV was confirmed in five plants from commercial fields and in all the plants from Zagreb. Due to the presence of single infections in plants from Zagreb, and in order to determine the detrimental effects of BYV, all plants from the study field control plots (104 of Torda and 103 of Libero) were tested by ELISA. Tests revealed the presence of BYV in 18 (17.3%) plants of Torda and in 34 (33%) plants of Libero. BYV infections averaged a 9 to 10% reduction of pure root yield and sugar content in both cultivars compared with BYV-free plants. One ELISA-positive plant per cultivar was chosen for the extraction of total RNA using the QIAGEN RNeasy plant mini kit according to manufacturer’s instructions. In both plants, the presence of BYV was confirmed by RT-PCR using four set of diagnostic primers (Kundu and Rysanek 2004). PCR products from both samples, named BYV- CRO-T from Torda and BYV-CRO-L from Libero, were purified and sequenced in both directions (Genbank Accession Nos. KP704263 to KP704268). Nucleotide sequence alignment of the DNA fragments for both Croatian isolates, including the C-terminal part of L-Pro and N-terminal part of MET and HSP70, showed 99.2, 99.7 and 100% similarity, respectively. Nucleotide comparisons of Croatian isolates with Californian (Genbank Accession No. AF056575) showed a 97% similarity for L-Pro and HSP70 and a 99% for MET. To our knowledge, this is the first report of Beet yellows virus in Croatia and its detrimental effect on actual sugar beet cultivars grown under Croatian environmental conditions.