First report of Tomato brown rugose fruit virus 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.

Association of 'Candidatus Liberibacter solanacearum' with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection.

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

Genetic structure and evolution of natural populations of viruses causing the tomato yellow leaf curl disease in Spain.

The population structure and genetic variation of two begomoviruses: tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl virus (TYLCV) in tomato crops of Spain were studied from 1997 until 2001. Restriction digestion of a genomic region comprised of the CP coat protein gene (CPR) of 358 TYLC virus isolates enabled us to classify them into 14 haplotypes. Nucleotide sequences of two genomic regions: CPR, and the surrounding intergenic region (SIR) were determined for at least two isolates per haplotype. SIR was more variable than CPR and showed multiple recombination events whereas no recombination was detected within CPR. In all geographic regions except Murcia, the population was, or evolved to be composed of one predominant haplotype with a low genetic diversity (<0.0180). In Murcia, two successive changes of the predominant haplotype were observed in the best studied population. Phylogenetic analysis showed that the TYLCSV sequences determined clustered with sequences obtained from the GenBank of other TYLCSV Spanish isolates which were clearly separated from TYLCSV Italian isolates. Most of our TYLCV sequences were similar to those of isolates from Japan and Portugal, and the sequences obtained from TYLCV isolates from the Canary island of Lanzarote were similar to those of Caribbean TYLCV isolates.