First report of tobacco necrosis virus causing the ABC-disease on potato tubers in Bulgaria

Potato (Solanum tuberosum) is a widely spread species and its cultivation is mainly concentrated in small to middle acreage (up to 5 ha) farms in Bulgaria. At the end of 2015, ABC-disease resembling symptoms (Noordam 1957) were found on freshly stored potato tubers in the region of Varna, in northeastern part of the country. About 5% of the tubers (cv. Melody) were damaged and possessed varying sized sections with (A) dark brown raised patches, (B) dark sunken lesions, and (C) light brown cracked patches. When a portion of symptomatic potato tubers was placed in a fridge at 3-4°C in darkness, the necrotic lesions became darker but no other or supplementary symptoms had developed after nine months storage. The ABC-disease and its pathogen tobacco necrosis virus (TNV) was found in Europe, firstly some decades ago in the Netherlands (Noordam 1957), then in Italy (Peters 1981), and most recently in Sweden (Beucha 2014). Symptomatic tubers were subjected to a DAS-ELISA screening using the RT-0070 antibody set detecting both A and D strains, including two positive controls, PV-0197 (bell pepper isolate) and PV-0415 (both from DSMZ GmbH, Braunschweig, Germany), healthy potato controls and blanks (no matrix). Positive reactions were only obtained in in the symptomatic samples and the positive controls (OD 405nm, Multiskan FC ThermoFisher Scientific, USA). Total RNA of two symptomatic tubers was extracted using the Spectrum™ Plant Total RNA Kit (Sigma Aldrich, Overijse, Belgium), pooled, and after passing quality control (QC; Nanodrop, bleach gel electrophoresis (assessing 18S & 28S band), and bioanalyzer (evaluating RNA Integrity Number (RIN) values)), the samples were sent for library preparation (NEBNext Ultra RNA library kit; New England BioLabs, Ipswich, MA, USA) and high throughput sequencing (Illumina NextSeq v2, small RNA sequencing; 3M single reads (1 x 50bp)) by Admera Health (NJ, USA). The sequence reads were quality filtered and submitted to the automated VirusDetect pipeline (Zheng et al. 2017). Mapping the resulting 1.24M reads against the viral database resulted in 24 viral contigs that only mapped against the reference strains; tobacco necrosis virus A (GenBank Accession No. M33002; 11 contigs (44-100bp), 41.7% coverage; 97.99% identity) and Nebraska (L04261; 13 contigs (41-115bp), 20.7% coverage, 95.93% identity). There were no hits with other viruses in the database. Additional direct mapping of the reads against reference genomes of tobacco necrosis virus strains using the SearchSmallRNA software (de Andrade and Vaslin 2014) revealed a genome-wide unique distribution of the mapped reads with the genomes of TNV-A isolates (AY546104, M33002, GQ221829 (Valence)) present in GenBank. About 65% of the TNV-A genome could be recovered through direct mapping against GenBank reference genomes. The smallRNA sequencing strategy resulted in a good coverage (79 %) for the RNA-dependent RNA polymerase open reading frame (ORF). Almost no contigs were recovered from the coat protein (CP) region, which is translated from subgenomic RNAs. No TNV satellite was detected and only a poor correspondence with TNV-D isolates was obtained. To the best of our knowledge, this is the first report of TNV on potato tubers in Bulgaria. Because of the widespread occurrence of its vector Olpidium brassicae special attention, at national level, needs to be paid to monitoring the spread of TNV to other hosts.

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