Virus Surveys in Olive Orchards in Greece Identify Olive Virus T, a Novel Member of the Genus Tepovirus

Forty samples representing 14 native Albanian and two foreign olive varieties were collected from an olive varietal collection plot in the Valias region (Tirana, Albania). The samples were assayed by RT-PCR for presence of olive-infecting viruses, including arabis mosaic virus (ArMV), cherry leaf roll virus (CLRV), cucumber mosaic virus (CMV), olive latent ringspot virus (OLRSV), olive latent virus 1 (OLV-1), olive leaf yellowing-associated virus (OLYaV), strawberry latent ringspot virus (SLRSV) and by PCR for the bacterium Xylella fastidiosa (Xf). Ninety-eight percent of the samples were infected with at least one virus. OLYaV was the most prevalent (85% of samples), followed by OLV-1 (50%), OLRSV (48%), CMV (28%), SLRSV (3%) and CLRV (5%), whereas ArMV and Xf were absent. Fifty-five percent of the samples were infected with one virus, 13% with two viruses, 20% with three, and 5% with four. Analyses of the nucleotide sequences of the Albanian virus isolates generally showed low genetic variability, and that most were phylogenetically related to Mediterranean isolates, in particular to those from Greece and Italy. Five olive trees, representing three native cultivars (‘Managiel’, ‘Kalinjot’ and ‘Kushan-Preze’) and one foreign (‘Leccino’), were found to be plants of the Conformitas Agraria Communitatis (“CAC”) category i.e. free of ArMV, CLRV, SLRSV and OLYaV. Only one tree of the native cultivar ‘Ulliri i kuq’ was free of all tested viruses, so this is plant material of the “Virus-tested” category. Olives derived from both categories could be used for propagation of standard quality plant materiel in a future certification programme for olive in Albania. This is the first report of CLRV, OLRSV, CMV and OLV-1 in Albania. The study also reveals the precarious health status of native olive varieties in the Valias varietal collection plot. However, the discovery of six plants representing two certifiable categories is a first step in a future olive tree certification program in the country.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Abstract Seed transmission of CLRV is a threat to gene bank contamination. CLRV-contaminated vegetative propagation material and seeds are prone to extremely long-distance transport. Due to this risk potential, CLRV is included in the list of plant viruses that should be closely monitored during sanitary production of propagative material, especially for walnut and olive trees (Bassi and Martelli, 2003). CLRV is treated as an A2 quarantine pathogen in Rubus in the EPPO region, a virus-free certification scheme for Rubus was developed by OEPP/EPPO (1994). In cherry (Kegler et al., 1972; Bush, 2005), walnut (Mircetich et al., 1980; Delbos et al., 1983; Nemeth et al., 1990) and olive production areas, CLRV infections are consistently occurring (Langer et al., 2010; Büttner et al., 2011); crop losses due to CLRV infections were reported for cherry (Kegler et al., 1972; Bush, 2005), walnut (Mircetich et al., 1980; Delbos et al., 1983; Nemeth et al., 1990) and raspberry (Jones and Wood, 1978).In birch species native to Fennoscandia virus-like symptoms on leaves (vein banding, leaf roll, mottling), partially adherent with progressive loss of vitality or death of twigs and branches have been spreading rapidly since 2002 with up to 85% of the tested trees being infected with CLRV (Jalkanen et al., 2007; von Bargen et al., 2009a).

Rhabdoviruses infect a large number of plant species and cause significant crop diseases. They have a negative-sense, single-stranded unsegmented or bisegmented RNA genome. The number of plant-associated rhabdovirid sequences has grown in the last few years in concert with the extensive use of high-throughput sequencing platforms. Here, we report the discovery of 27 novel rhabdovirus genomes associated with 25 different host plant species and one insect, which were hidden in public databases. These viral sequences were identified through homology searches in more than 3000 plant and insect transcriptomes from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) using known plant rhabdovirus sequences as the query. The identification, assembly and curation of raw SRA reads resulted in sixteen viral genome sequences with full-length coding regions and ten partial genomes. Highlights of the obtained sequences include viruses with unique and novel genome organizations among known plant rhabdoviruses. Phylogenetic analysis showed that thirteen of the novel viruses were related to cytorhabdoviruses, one to alphanucleorhabdoviruses, five to betanucleorhabdoviruses, one to dichorhaviruses and seven to varicosaviruses. These findings resulted in the most complete phylogeny of plant rhabdoviruses to date and shed new light on the phylogenetic relationships and evolutionary landscape of this group of plant viruses. Furthermore, this study provided additional evidence for the complexity and diversity of plant rhabdovirus genomes and demonstrated that analyzing SRA public data provides an invaluable tool to accelerate virus discovery, gain evolutionary insights and refine virus taxonomy.

A one-step reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed and used for the detection of <i>Cherry leaf roll virus</i> (CLRV) and <i>Strawberry latent ring spot virus</i> (SLRSV). The protocol was used to test infected screen house plants and also plants from orchards and vineyards where the vector (<i>Xiphinema diversicaudatum</i>) of SLRSV was detected from the soil. The one-step RT-PCR protocol is rapid and sensitive and has the potential to be used for the diagnosis of CLRV and SLRSV in routine diagnostic laboratories.