First report of cucumber mosaic virus infecting Azorina vidalii in Azores Islands

Virus diseases that occur in crops pose a major threat not only to global food security but also to wild plant communities growing in natural ecosystems (Jones, 2020, and references therein). In Azores (Portugal) little is known about viruses present on native flora and therefore, they have not been taken into consideration in conservation programs. Considering this, we selected Azorina vidalii (Campanulaceae), an endangered (IUCN) plant, endemic to Azores (Bilz, 2011), to survey for plant viruses. A. vidalii, the sole species of its genus, is often found in crevices with no soil accumulation on coastal cliffs, exposed to storms and sea spray, and is used as an ornamental. Leaves from 53 plants of A. vidalii from three populations from Terceira Island and three populations from Flores Island were randomly collected without obvious symptoms of virus infection, between the summer of 2021 and fall of 2022. RNA extraction was performed using the Plant/Fungi Total RNA Purification Kit (Norgen Biotek, Canada). RNA extracts from each population were pooled into six distinct composite samples (AvT1, AvT2, AvT3, AvF1, AvF4 and AvF5) and sent to Lexogen (Austria) for small RNA library preparation and High-Throughput Sequencing. Single-end RNA sequencing using Illumina NextSeq2000 system yielded between 10.1 M and 33.8 M raw reads. Adaptors and low-quality reads were removed with Trim Galore! and PRINSEQ. Trimmed reads were mapped to the genome phylogenetically nearest to A. vidalii available at the NCBI database (Adenophora triphylla). The resulting 2.5 M - 13.5 M unmapped reads were analysed with VirusDetect online version (database v248) (Zheng et al., 2017) for virus detection and identification. Sequences of cucumber mosaic virus (CMV) (contigs of up to 3045 nt for RNA1, 2917 nt for RNA2 and 2086 nt for RNA3) were identified in five (AvT1, AvT2, AvT3, AvF1 and AvF5) of the six composite samples and CMV satellite sequences (two contigs with 145 and 197 nt) were identified in only one (AvT1) of the composite samples. To confirm the presence of CMV, all samples were tested by two-step RT-PCR using primers targeting CMV-specific RdRp gene (513 bp) (Grieco et al., 2000), which retrieved 18 positive samples (34%). Nine samples were selected for Sanger sequencing (six out of 13 from Terceira and three out of five from Flores) based on digestion profile obtained with AluI and MboI. The resulting sequences (OQ176229-OQ176233, OQ732757-OQ732760) share an identity of 97.2-100% and BLASTn showed them to have 98.3-99.6% identity to CMV strain TN (AB176848). A Neighbour-Joining tree (Supplementary material) inferred in MEGA11 (Tamura et al., 2021) with 237 additional CMV-RdRp sequences, showed that A. vidalii CMV-derived isolates clustered together with reference strains of subgroup II, as those used by Roossinck (2002) for phylogenetic analysis of the 2a ORF. Besides CMV, tomato spotted wilt virus and polerovirus-associated RNAs sequences were found in one of the A. vidalii populations, but with lower coverage, and need to be further investigated. To the best of our knowledge, this is the first report of CMV infecting A. vidalli. CMV, genus Cucumovirus, is an agriculturally important virus and one of the most successful viruses known, infecting over 1,200 species of plants (Palukaitis & García-Arenal, 2003). In addition to A. vidalii being a CMV reservoir, which may have implications on adjacent crop fields, further research is needed to investigate the impact of CMV on A. vidalli fitness.