First report on the occurrence of Little cherry virus 1 in Romania

Little cherry disease (LChD) is considered an economically important viral disease of sweet cherry. It is associated with two different members of the family Closteroviridae, Little cherry virus 1 (LChV-1, genus Velarivirus) and Little cherry virus 2 (LChV-2, genus Ampelovirus). While its presence was recorded from most part of the world, uncertainties and extremely limited information are available about its occurrence in Romania. LChV-1 is known to be transmissible by grafting and is spread via infected propagated plant material. No vector has been identified yet. Similar pathways of transmission are known for LChV-2. In addition, LChV-2 can be transmitted by at least by two species of mealybugs (Hemiptera, Pseudococcidae). During the growing season of 2017, a first survey was performed in two old sweet cherry orchards located in Bistrița area from Southern Romania. Based on LChD typical symptoms expression especially early leaf reddening, two trees from an orchard were selected for further molecular detection. Leaves samples (fresh and lyophilized) and shoots were collected and used for total RNA extraction. cDNA was prepared from tenfold diluted total RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Belgium) according to the manufacturer’s instructions. RT-PCR detection of LChV-1 was carried out using previously described specific primers: LCUW7090 (5′-GGTTGTCCTCGGTTGATTAC-3′) / LCUWc7389 (5′-GGCTTGGTTCCATACATCTC-3′) (Bajet et al., 2008), amplifying a 300-bp fragment spanning the LChV-1 ORF1b encoding the RNA dependent RNA-polymerase (RdRp) gene. Samples from both orchards showed positive results for LChV-1. No LChV-2 was detected using LC26L/LC26R primer pair (Eastwell and Bernardy, 2001). Bidirectional sequencing of RT-PCR products was carried out (Macrogen Inc., Amsterdam, The Netherlands) and BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for the sequenced genomic regions was performed. BLAST analysis of the LChV-1 RdRp sequences from Romania revealed 94% similarity amongst isolates and 95% and 98% identity with sequences from Greek isolates (HG792394 and HG792418, respectively). Our preliminary finding represents an important step in this investigation which will be supported by more molecular characterization and phylogeny. These results will be useful in the differentiation and analysis of closely related LChV-1 viral isolates and are extremely important for LChD epidemiological studies towards field disease control and management guidelines to prevent spread to Prunus germplasm at regional and national level. To our knowledge this is the first report of molecular detection of LChV-1 in Romania.