First Report of Tomato Brown Rugose Fruit Virus Infecting Tomato in Greece

Abstract

During summer and autumn 2019, virus-like disease symptoms were observed in tomato (Solanum lycopersicum) grown in several greenhouses in Chania (Crete, Greece) and Kyparissia (Messenia, Peloponnese, Greece). The disease showed a particularly aggressive profile reaching in some cases 100% incidence. Symptoms included mild to severe mosaic, deformations, and blistering on leaves accompanied by brown and yellow spots on fruits. Sap inoculation of extracts from symptomatic tissues derived from seven tomato plants on Nicotiana tabacum cv. Xanthi-nc plants resulted in local necrotic lesions, indicative of a tobamovirus infection. To identify the virus, total RNAs were extracted from the seven symptomatic tomato plants and examined with a commercial tobamovirus screen reverse transcription PCR (RT-PCR) kit (LOEWE Biochemica, Germany). PCR products of 568 nucleotides were obtained from all samples examined. The seven PCR products were further subjected to sequencing. BLAST analysis of the sequences revealed the presence of tomato brown rugose fruit virus (ToBRFV) in all samples (sharing 100% nucleotide identity with each other and 99% identity with GenBank accession MK881102 from Palestine). ToBRFV, a relatively new member of the genus Tobamovirus, was originally described in Jordan (Salem et al. 2016). It is already known for its ability to overcome the so far well-established Tm-2² resistance to tobamoviruses in tomato. To further characterize the Greek isolate, total RNA from an infected tomato plant was subjected to Illumina RNA-Seq. The analysis of approximately 14,000,000, 75-bp single-end reads confirmed the presence of ToBRFV (and the absence of other viruses in the sample). BLAST analysis of the assembled sequence of the Greek isolate (ToBRFV-Gr) (6,354 nucleotides, GenBank accession no. MN815773) revealed a close relationship to the Israeli ToBRFV-IL isolate (99.8% identity, GenBank accession no. KX619418). The ToBRFV-Gr isolate was propagated through mechanical inoculation in N. tabacum ‘Turkish Samsun’, and infected tissue from the latter was used to reproduce symptoms in virus tested-negative tomato plants (cv. Rutgers). Ten days later the inoculated plants showed narrowed leaves with mild mosaic symptoms similar to those described previously by Luria et al. (2017). The presence of ToBRFV was confirmed in symptomatic leaves by RT-PCR using primer pairs ToBRFVGr-F (5′-GAATATCCGGCCTTGCAGAC-3′) and ToBRFVGr-R (5′-TCGAAATTCCACATAAGGTTGGC-3′) and by double antibody sandwich ELISA with the commercial polyclonal antibodies against ToBRFV (LOEWE Biochemica). Τhis is the first report of ToBRFV in Greece. The fact that the virus can be easily transmitted mechanically (through wounding, human activities, contact with infected plants, and insects used for pollination) and by infected seeds, in combination with the lack of any commercial resistant tomato cultivars, constitutes an emerging threat to Greek tomato and potentially pepper cultivation. ToBRFV is included in EPPO’s alert list and is regulated in the European Union since November 2019 (Commission Implementing Decision [EU] 2019/1615). Containment actions against the further spread of ToBRFV were taken at the sites of its emergence.