First Report of Apple Luteovirus 1 in Apple Trees in Greece

Abstract

In June 2018, shoots were collected from five apple trees from different orchards in Zagora (Magnesia prefecture, east-central Greece) exhibiting various virus-like disease symptoms such as leaf mosaic and bark scaling or suffering from apple small fruit disorder. To identify the agents associated with the symptomatology, total RNAs were extracted following a CTAB protocol (Gambino et al. 2008), and after DNase treatment, equimolar quantities from each of the five extracts were mixed in a pooled sample and subjected to high-throughput sequencing (HTS) after ribosomal RNA was removed using a Ribo-Zero rRNA Removal Kit (Plant Leaf, Illumina). Library preparation followed by sequencing on an Illumina NextSeq Sequencing System (Biomedical Research Foundation Academy of Athens, Greece) resulted in 55 million 75-bp single-end RNA sequencing reads. The analysis of the HTS data with Geneious 11.1.5 software revealed the presence of viruses frequently found in apple cultivation in Greece (apple chlorotic leaf spot virus, apple mosaic virus, apple stem pitting virus, apple stem grooving virus), but also the presence of the recently discovered apple luteovirus 1 (ALV1) (Liu et al. 2018). In total, 316,984 reads were de novo assembled in one viral contig annotated by BLAST as ALV1 sequence, covering 99% (devoid of untranslated regions) of the genome of the reference isolate PA8 (GenBank accession no. NC_040680) and sharing 92.3% nucleotide identity. Based on the HTS analysis, primers were designed (LUTEO-Forward, 5′-GCCAGCTGAGGTCTTCACTT-3′; LUTEO-Reverse, 5′-GAAGTCGAATACCAACGCGC-3′) to amplify part (674 bp) of the ORF3 to ORF5 genomic regions of the virus. Subsequently, the five original trees and 12 more with similar symptoms from the same region were tested with reverse transcription PCR using the new pair of primers. PCR amplicons of the expected size were obtained in four of the five trees subjected to HTS, and in 11 of the other 12 trees examined. Two of the PCR products were subjected to Sanger sequencing (100% identity with each other, GenBank accession no. MT068445), confirming the presence of ALV1 (98.4% nucleotide identity with isolate PA8). The virus was first described in apple trees suffering from “rapid apple decline” in the Northeastern United States (Liu et al. 2018), but it was not found in declining trees in Washington State (Wright et al. 2020). It was also detected in apple rootstocks showing yellowing and mottling symptoms in Korea (Lim et al. 2019). In all cases, including the present study, the virus was found in mixed infections with other viruses, making difficult its association with a specific symptomatology. Nevertheless, our results suggest that this recently discovered virus is widespread in Zagora, one historical apple-producing area in mountainous Greece with many old orchards, a fact that can increase our knowledge on the diversity and possible pathogenicity of ALV1. Moreover, due to its proposed association with rapid apple decline, it constitutes a possible threat for apple cultivation in Greece. To the best of our knowledge, this is the first report of ALV1 in apple trees in Greece and generally in Europe.