Characterization of the virome of ancient pome fruit cultivars of Malus Mill. and Pyrus L. using high-throughput sequencing

Abstract

Pome fruit viruses have been mainly identified from commercialised cultivars presenting disease symptoms. Their significant impact on fruit yield and quality triggered their identification and characterization ex post. For example, the infection of certain virulent strains of apple stem pitting virus (ASPV) was detected due to necrosis development between susceptible scions and/or rootstocks (rootstock incompatibility). In this context, the diversity of viruses infecting pome fruit trees is still largely underestimated and it is worth additional investigation. The goal of this project is to evaluate the presence of known and unknown viruses infecting ancient cultivars of apple and pear. Leaf samples were taken from six apple cultivars (‘Gravenstein’, ‘Pomme Pellone’, ‘Délices de Beignée’, ‘Reinette Meurens’, ‘Belle de Boskoop’, and ‘Joseph Musch’) and five pear cultivars (‘Poire Cuisse Madame’, ‘Jeanne d’Arc’, ‘Poire Rougette’, ‘Bronzée d’Enghien’, and ‘Colmar du Mortier’). Total RNA was analysed from ‘Joseph Musch’ (tree Q9) after high throughput sequencing. Double-stranded RNA (dsRNA) and virion-associated nucleic acids (VANA) preparation protocols were applied on the other samples individually (dsRNA) or pooled (dsRNA and VANA). After sequencing, the obtained data were analysed with Geneious Prime to identify the viruses present in the sampled trees and to reconstruct their genomes. In addition, two other bioinformatics pipelines were tested on the generated data (Kaiju and Kraken). The nearly complete genome sequence of seven new isolates from several known viruses was reconstructed. All the samples were infected by at least one virus, the most prevalent was ASPV. Interestingly, the detection of Apple rubbery wood virus-1 (ARWV-1), Apple luteovirus-1 (ALV-1) and Apple hammerhead viroid-like RNA (AHVd-like RNA) corresponded to the first detection in Europe. Primer and RT-PCR protocols were designed. The presence of ARWV-1 was detected by RT-PCR, confirming the first detection of this virus in Europe. Further studies need to be carried out to assess the distribution of ARWV-1 within the germplasm collection and to confirm the detection of ALV-1 and AHVd. The analysis of the local prevalence of these viruses will be a first step to evaluate the biological risk they can pose for European production.