Introduction Host range is an important aspect of the viral Little cherry disease (LChD). LChD can be caused by distinct viruses (Little cherry virus 1 and 2, Closteroviridae), and is a major constraint to sweet and sour cherry (Prunus avium L. and P. cerasus L.) production worldwide (3). LChV-1 (genus Velarivirus), is known to be graft-transmissible, and is spread with infected propagation plant material, but no vector is known so far. For LChV-2 (genus Ampelovirus) at least two distinct species of mealybugs (Phenacoccus aceris and Pseudococcus maritimus, Hemiptera, Pseudococcidae) have been known to transmit the virus. In this study, we characterize novel LChV-1 plum (P. domestica L. cv. Opal) isolates collected in trees growing at the edge of a plum orchard in the vicinity of a sweet cherry (P. avium L.) orchard known to be infected with LChV-1. Materials and Methods Total RNA of leaves and roots collected from plum trees was extracted using the Spectrum Total Plant RNA kit (Sigma-Aldrich, Belgium) and diagnostically tested by RT-PCR with LChV-1 specific primers amplifying major genomic ORFs (1, 2). Subsequently to bidirectional amplicon sequencing, sequences were assembled using BioNumerics V7.6.1 and were compared in GenBank database using BLASTn. The evolutionary relationships of LChV isolates were reconstructed based on partial genomic nucleotide sequences using Maximum likelihood phylogenetic trees inference in MEGA6. Results and Discussion From an epidemiological point of view, the presence of LChV-1 on P. domestica L. was confirmed for the first time in Belgium in 12% of the plum samples. BLAST of the assembled amplified sequences revealed a distinct molecular variability between the plum and cherry isolates with significant divergence between the sequences of both adjacent respective orchards suggesting separate viral introductions. Based on RdRp gene sequences, the Belgian plum isolates shared highest identity with the Greek cherry (HG792418) and peach isolates (HG792399) while the Belgian cherry ...