First report of in tomato in Spain.

In October 2019, tomato (Solanum lycopersicum L.) plants showing chlorosis and brown necrosis in apical leaflets and rugose surface in fruits were observed in a greenhouse in Vicar, Almería, Spain. A total of 0.5% of the tomato plants in the greenhouse (1,38 ha) showed these symptoms. The presence of tomato brown rugose fruit virus (ToBRFV) was suspected. A total of 5 symptomatic and 2 symptomless leaf samples were collected and analyzed by double-antibody sandwich (DAS)-ELISA with antibodies for ToBRFV, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) (Loewe Biochemica, Germany). Symptomatic samples tested positive by DAS-ELISA only for ToBRFV. Therefore, one sample was selected and analyzed by reverse transcription (RT)-PCR with specific primers ToBRFV-F5722/ToBRFV-R6179 for ToBRFV (Panno et al. 2019a) which amplified a 458 bp fragment of the coat protein gene. The sequence obtained by Sanger sequencing from the amplicon showed 99.7% nt identity with ToBRFV isolate from United Kingdom (Acc. No. MN182533) and was deposited in the GenBank database under the accession number MT211630. Further surveys were performed in Vicar and El Egido (Almería, Spain) on plants showing viral-like symptoms. A total of 50 tomato and two pepper leaf samples from 28 greenhouses were collected and analyzed by DAS-ELISA for ToBRFV, TMV, ToMV, pepino mosaic virus (PepMV) , tomato spotted wilt virus (TSWV) and tomato yellow leaf curl virus (TYLCV) with respective antibodies (Loewe Biochemica, Germany). Five tomato plants (four from Vicar and one from El Ejido) tested positive for ToBRFV. PepMV, ToMV and TYLCV were also detected in one, three and five tomato plants, respectively. The ELISA positive results for ToBRFV were confirmed by end point RT-PCR with primer pairs ToBRFV-F5722/ToBRFV-R6179 and ToBRFV-F/ToBRFV-R (Alkouni et al., 2019) and by real-time RT-PCR with Taqman probe ToB-probe and specific primers ToB5520F/ToB5598R (Panno et al., 2019b). Tomato seeds used for plantation in these greenhouses were also analyzed, but ToBRFV was not detected. Eradication measures have been undertaken to prevent the virus spread and to control this outbreak. Official seed analysis by DAS-ELISA and real time RT-PCR (ISF, 2019) are being conducted on the tomato and pepper imported seeds to prevent the appearance of new sources of ToBRFV inoculum in Spain. References: Alkowni, R., et al. 2019. J. Plant Pathol. 101: 719. doi: 10.1007/s42161-019-00240-7. ISF, 2019. International Seed Federation. Version 1.3, September 2019. Available at https://www.worldseed.org/wp-content/uploads/2019/09/Tomato-ToBRFV_2019.09.pdf Panno, S., et al. 2019a. Plant Dis. 103: 1443. https://doi.org/10.1094/PDIS-12-18-2254-PDN Panno, S. et al. 2019b. PeerJ 7:e7928 DOI 10.7717/peerj.7928.