First report of strawberry polerovirus 1 in strawberry in Italy.

Abstract

Strawberry (Fragaria × ananassa Duch.) was introduced in Sicily (Italy) in the 1930s in the small town of Maletto, on the slopes of Etna volcan, where it's currently cultivated in a total area of 30 ha. The French cv. 'Madame Moutot', appreciated for its unique flavor and intense fragrance, was there propagated vegetatively and after decades, the distinctive 'Etna ecotype' originated by adaptation to the peculiar environmental conditions of the area (Milella et al., 2006). In May 2023, in a 0.5 ha "Etna ecotype" strawberry field, virus-like symptoms were observed in approximately 50% of the plants. Symptoms included severe dwarfing, leaf cupping and chlorotic spotting which lead to decline of infected plants. To investigate the etiology of the disease, leaf samples were collected from eight symptomatic plants for analysis by High-Throughput Sequencing (HTS). To this aim, total RNAs were extracted by using the RNeasy PowerPlant Kit (Qiagen, Germany). The RNAs were pooled, depleted of ribosomal RNA (QIAseq FastSelect; Qiagen), and a library was prepared according to the Illumina DNA Prep Kit. Sequencing on a NextSeq2000 instrument at Leibniz Institute DSMZ (Braunschweig, Germany) generated 31,149,784 of paired-end reads (150 nt), which were further analyzed in Geneious Prime version 2023.2 (Biomatters) using a custom workflow for virus discovery and genome assembly. Analysis of the assembled contigs by local BLASTn and BLASTp alignments against a custom plant virus database of NCBI nuclear-core (NC) reference sequences assigned a number of contigs to accession NC_025435, strawberry polerovirus 1 (SPV-1). Reconstruction of the virus genome by assembly of contigs and reads alignment resulted in a nearly complete genome sequence of SPV-1 (GenBank Acc. No. OR989958) showing by BLASTn 98.69% identity to the SPV-1 NC reference sequence, and 98.99 % identity with an isolate from the Czech Republic (GenBank Acc. OL421571). To confirm the presence of SPV-1 in each sample, RT-PCR using specific primers designed in this study SPV-1-CP-1F (5'-TCGAGATACGTCTAGAACTGCAA-3') and SPV-1-CP-1R (5'-GAGAGGCCCCTTCTACCTATTTG-3') targeting the entire 623 bp coat protein (CP) gene was performed. Amplicons of the expected size were obtained in five samples and Sanger-sequenced. The resulting sequences shared 99.85% - 100% of identity to the HTS - derived sequence (GenBank Acc. No. OR989958) through BLASTn analysis. Strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV) and strawberry crinkle virus (SCV) were detected in the same library in addition to SPV-1 and then confirmed by RT-PCR using specific primers (Martin & Tzanetakis 2013). Strawberry polerovirus 1, related to the genus Polerovirus in the family Solemoviridae, was first reported in strawberries in Canada (Xiang et al. 2015) and was thereafter detected in the United States (Thekke-Veetil & Tzanetakis 2016), Argentina (Luciani et al. 2016), and Nepal (Kuwak et al. 2022). To date, the virus has been reported in Europe only in the Czech Republic (Franova et al. 2021). To our knowledge, this is the first report of SPV-1 in strawberry plants in Italy. Although the correlation between SPV-1 and strawberry decline (SD) is still uncertain (Xiang et al. 2015) transmission of the virus via aphids has recently been demonstrated (Franova et al. 2021). Our report let to hypothesize that its dissemination in Europe can be considered as increasing.