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High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data

A disease of tomato (Lycopersicon esculentum Mill.) was observed in three greenhouses in Tömörkény in southern Hungary in the autumn of 2007. Thirty percent of the plants were chlorotic and stunted and had mottled leaves with interveinal yellowing and necrosis. Similar symptoms induced by Tomato chlorosis virus (ToCV) on tomato have been reported in other countries (1,2). ToCV is a Crinivirus in the Closteroviridae family, which can cause a decline in plant vigor and reduced fruit yield. ToCV is transmitted by whiteflies (Trialeurodes vaporariorum West., T. abutilonea Hald., and Bemisia tabaci Genn.) and grafting, but cannot be transmitted mechanically. Only T. vaporariorum is known to be present and widespread in Hungary. Virus presence was confirmed by reverse transcription-PCR as described by Louro et al. (2). cDNA synthesis with ToCV specific primers (ToCV-UP 5′-TCATTAAAACTCAATGGGACCGAG-3′ (3) and ToCV-DW 5′-GCGACGTAAATTGAAACCC-3′) was successful and electron microscopy revealed ToCV-like particles. The PCR product has been sequenced (GenBank Accession No. HQ444266) and showed 97 to 99% identity to ToCV isolates in GenBank. According to the symptoms, amplified region, sequence data, and electron microscopy, the tomato samples from Tömörkény were confirmed to be infected with ToCV. The economic losses associated with ToCV were minor. To our knowledge, this is the first report on the occurrence of ToCV in Hungary. References: (1) G. P. Accotto et al. Plant Dis. 85:1208, 2001. (2) D. Louro et al. Eur. J. Plant Pathol. 106:589, 2000 (3) J. Th. J. Verhoven et al. Plant Dis. 87:872, 2003.