First Report of Cherry virus A and Plum bark necrosis stem pitting-associated virus in Cherry in Chile

Stone fruits rank third among the most important crop species in Chile, after grapevine and apple. Specifically, cherry (Prunus avium L.) cultivation have increased during the last 10 years, making of Chile the most important exporter in the Southern hemisphere. Nineteen cherry samples collected in the spring of 2016 were subjected to high-throughput sequencing (HTS) analyses. Small RNA extracts were obtained following the protocol described by Giampetruzzi et al. (2012). Sequencing libraries were prepared using TruSeq smallRNA library preparation kit (Illumina Inc.) and sequenced on Illumina HiScanSQ platform. Trimming and de novo assembly of sequenced reads using CLC genomics workbench v7.0 were carried out, and the obtained contigs were analyzed with BLAST. One sample presented 131 contigs that showed homology with the Plum bark necrosis stem pitting-associated virus (PBNSPaV) reference sequence with accession no. EF546442. The alignment of nine PBNSPaV complete genome references allowed the design of two primer pairs specific for the RdRp gene (PBN-RdRp-F 5?-CTTATTATTGTGCTGAAGTTGATCT-3?/PBN-RdRp-R 5?-TGGAAAAGTATTGAGTCATCACC-3?) and a partial region of CP gene (PBN-CP-F 5?-GAGGCAATGGATGAGGAA-3?/PBN-CP-R 5?-TCTTCCACCGGACTGATTA-3?) to be used in RT-PCR. The RdRp (KY887573) and CP (KY887574) sequences amplified from isolate 10381 shared 99 and 97% identity with reference isolate WH1 (KJ792852) from China and the PBNSPaV type-strain (EF546442) from the U.S.A., respectively. In addition, the HTS analysis showed that 14 out of 19 cherry samples have several contigs showing homology with Cherry virus A (CVA) reference sequence. Two primer pairs (CVAF1 5?-CAATGTTGTTGACAATTCCCAC-3?/CVAR1 5?-CCTACATGAATTTGACCTAAACAAA-3?; CVAF2 5?-ACTGCAGAGAAAACAACTGCC-3?/CVAR2 5?-AGGCCCCTTCTTATCTCGTT-3?) were designed based on the alignment of CVA complete genomes database with the sequences obtained from Chilean isolates. CVA infection was confirmed via RT-PCR in all 14 cherry trees using both primer pairs. BLASTn analysis of the two amplification products of CVA from isolate 10596 (KY887575, KY887577) showed 99% of identity with the isolate Lambert (KU215410) from Czech Republic and the same amplicons obtained from isolate 10395 (KY887576, KY887578) showed 99% of identity with the isolate ChTA12 from China (KT310083). To our knowledge, this is the first report of CVA and PBNSPaV infecting cherry in Chile and South America. Further analyses are in progress in order to determine the prevalence of these viruses in the main cherry producing areas of Chile.