Three autochthonous grapevine varieties of Majorca (Spain) were analyzed for the presence of viruses listed by the international certification programs. Enzyme-Linked Immuno-Sorbent Assay (ELISA) screenings were performed in 193 vines from 46 vineyards included in a clonal selection. Virus-free vines were only 6.4%, 9.6% and 11.5%, in Manto Negro, Callet and Moll, respectively. Infections by grapevine leafroll associated viruses (GLRaVs) were ascertained in 71%, 78% and 60% of Manto Negro, Callet and Moll vines, respectively. Each variety was also highly infected by Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV). The percentage of plants displaying multiple infections was 58.4% in Manto Negro, 63.8% in Callet and 42.6% in Moll. Thus, it was very difficult to identify virusfree clones with suitable agronomic characteristics to be considered as a reference for the grape market. In order to obtain certified propagation material under such conditions of endemic viral infection, sanitation should be the main focus in clonal selection processes. However, the time and financial requirements for proper sanitation process bring to consideration the need to use, at least temporarily, standard multiplication material while certified clones are achieved.Additional key words: autochthonous varieties, grapevine certification, standard material virus incidence. Resumen Incidencia de las infecciones víricas en antiguos viñedos de tres variedades de vid autóctonas de Mallorca: consecuencias sobre las estrategias de la selección clonalEn este estudio se analizó la presencia de las virosis contempladas por las leyes internacionales en tema de certificación en tres variedades de vid autóctonas de Mallorca. Para ello se realizó el test ELISA (enzyme-linked immunosorbent assay) sobre 193 cepas procedentes de 46 viñedos incluidos en un programa de selección clonal. Las cepas que resultaron libres de los virus analizados fueron el 6,4% en Manto Negro, el 9,6% en Callet y el 11,5% en Moll. Los porcentajes de cepas infectadas por los virus asociados al síndrome del enrollado de la vid (GLRaVs) fueron 71% en Manto Negro, 78% en Callet y 60% en Moll. Se detectaron también altas tasas de infecciones para el virus del entrenudo corto infeccioso de la vid (GFLV) y el virus del jaspeado de la vid (GFkV) en las tres variedades. Los porcentajes de cepas sujetas a infecciones múltiples fueron 58,4% en Manto Negro, 63,8% en Callet y 42,6% en Moll. Por tanto fue difícil encontrar cepas que cumpliesen tanto los requisitos sanitarios para la certificación como aquellos agronómicos necesarios según las exigencias del mercado vitivinícola. En tales condiciones de infecciones víricas endémicas, el saneamiento debería ser el primer objetivo a seguir con el fin de obtener material de propagación certificado. Sin embargo, el tiempo y los recursos económicos necesarios para llevar a cabo el proceso de saneamiento conllevan la necesidad de considerar el uso temporal de material de propagación estándar hasta que se obtengan clones cer...

The effects of viruses on grape production and must quality are not fully understood. In this study, the evaluation of the impact of different virus infections on performance of the main autochthonous grapevine varieties of Mallorca (Callet, Manto Negro and Moll) was pursued. Therefore, a large number of vines were observed in field conditions over 4 years and tested by enzyme‐linked immunosorbent assay for viruses listed by the international certification programmes. In each variety, some specific virus infections resulted to be more effective than the others in inducing losses in production. In Callet, yield (Y) reduction was over 20% in plants infected by Grapevine fanleaf virus (GFLV). In Moll, plants subject to more than one infection showed over 40% Y decrease. In Manto Negro, the most surprising results were obtained, because plants showed almost 40% Y reduction because of Grapevine leafroll‐associated virus‐1 (GLRaV‐1) infection. In addition, virus infections were linked to some must quality parameter increase in Manto Negro and Moll, but in the majority of cases it was an indirect effect, because the decrease in production parameters played a predominant role by producing an important concentration effect. However, in Manto Negro, anthocyanin content decrease was directly related to GFLV infection.

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Biological indexing is the method generally recognized for the certification of propagative grapevines in many countries, and it is mandatory in the European Union. It consists of the evaluation of the plant material after grafting on indicators that are inspected for symptom development. This is a lengthy process that requires well-trained workers, testing field, etc. Alternative diagnostic methods such as serology and RT-qPCR have been discarded for certification because of their intrinsic drawbacks. In turn, high-throughput sequencing (HTS) of plant RNA has been proposed as a plausible alternative to bioassay, but before it is accepted, different aspects of this process must be evaluated. We have compared the HTS of small RNAs with bioassays and other diagnostic methods from a set of 40 grapevine plants submitted for certification. The results allowed the authors the identification of numerous grapevine viruses in the samples, as well as different variants. Besides, relationships between symptom expression and viromes were investigated, in particular leafroll-associated viruses. We compared HTS results using analytical and bioinformatics approaches in order to define minimum acceptable quality standards for certification schemes, resulting in a pipeline proposal. Finally, the comparison between HTS and bioassay resulted favorable for the former in terms of reliability, cost, and timing.

During phytosanitary certification of three grapevine clones (one ofGorgollosa (Osman et al., 2007) detected Grapevine leafroll-associated virus 4.Using similar real-time PCR methods we confirmed the presence of GLRaV-2 but we did not detect either GLRaV-1 or GLRaV-3. Moreover we could also detect Grapevine fanleaf virus, GFLV (DAS-ELISA, Bioreba) in the Mances de Tibus vines; this was confirmed by symptom inspection in the indicator host Rupestris de Lot.In order to confirm the presence of , RNA extracts were also tested by conventional RT-PCR with consensus primers derived from the GLRaV-4 hsp70 gene: and . Amplicons were of the expected size (238 bp) and were cloned using pGEMT-Easy (Promega).Comparisons showed that sequences were identical in all three isolates (GenBank Accession No. GU735409) and showed 99% nucleotide identity with the homologous hsp70 genes of GLRaV-4 isolates LS-24 from China (GQ849394) (Pei et al., 2010) and Cl-3642 from Chile (EU746619) (Escobar et al., 2008).

Grapevine leafroll-associated viruses (GLRaVs) cause significant reductions in yield and quality in the wine industry worldwide. At least nine different GLRaVs have been found in different regions of the world. In the process of virus indexing of candidate grapevine clones for certification, which includes grafting of scions onto rootstocks, we observed strong leafroll symptoms 1 year after grafting with one vine of cv. Estaladina in Castilla y León, Spain and one vine of cv. Tempranillo in La Rioja, Spain, collected in 2008 and 2007, respectively. Both vines tested positive by real-time reverse transcription (RT)-PCR with TaqMan probes specific for Grapevine leafroll-associated virus 5 and double-antibody sandwich (DAS)-ELISA with a mix of monoclonal antibodies that recognizes GLRaV-4, 5, 6, 7, and 9 (Bioreba, Reinach, Switzerland). RNA extracts of both GLRaV-5 positive vines were analyzed by conventional RT-PCR with a pair of consensus degenerated primers derived from GLRaV-5 hsp70 sequences available in GenBank: LR5HYF (5′-TGGGATGAAYAARTTCAATGC-3′) and LR5HYR (5′-TGAAATTCCTCATRTARGAGC-3′) that amplified a 250-bp fragment. Amplicons were cloned and the comparison of the amino acid sequences (Estaladina isolate, Est110: Accession No. HM208622; Tempranillo isolate, Tem020: Accession No. HM208618) showed in the case of the Est110 isolate, 100 and 82.6% identity, respectively, with the homologous genes of one GLRaV-5 isolate from the United States (AF233934 [3]) and Argentina (EU815935 [2]). For isolate Tem020, the hsp70 gene showed 97.1 and 81.2% amino acid identity with the homologous hsp70 genes of the United States and Argentina isolates. The coat protein (cp) genes of both isolates were also amplified and cloned using the specific GLRaV-5 primers, LR53413 (5′-CGTGATACAAGGTAGGACAACCGT-3′) and LR53843 (5′-CTTGCACTATCGCTGCCGTGAAT-3′), designed according to the sequence of AF233934. Fragments were of the expected size (430 bp) and the nucleotide sequences were obtained (Est110: Accession No. HM363522; Tem020: Accession No. HM363523) and used for pairwise nucleotide comparisons. The Est110 isolate showed 96.7 and 97.5% amino acid identity with the isolates from the United States and Argentina, respectively, while the Tem020 isolate showed 94.8 and 95.6% identity, respectively. Amino acid identity of Est110 and Tem020 cp genes was 100% when compared with the homologous genes of isolates AF233934 and EU815935. To our knowledge this is the first report of GRLaV-5 in Spain. Since 2008, we have detected eight additional vines positive for this virus in 200 clones analyzed for certification, suggesting that the incidence of GLRaV-5 in Spain could be widespread. This research indicates that virus indexing for GLRaV should be included in certification schemes for grapevine candidate clones (1) in Spain. References: (1) Anonymous. OEPP/EPPO Bull. 38:422, 2008. (2) S. Gomez Talquenca et al. Virus Genes 38:184, 2009. (3) F. Osman et al. J. Virol. Methods 141:22, 2007.

In Europe, many autochthonous grapevines (Vitis vinifera L.) are only cultivated at local scale, but play a very important economic role due to their strict relation with terroir and wine tipicity. In this study, it was pursued to evaluate the influence of several factors on performance of Moll, the main autochthonous white variety of Majorca, by means of the data-

Andalusia is a Spanish region that is home to numerous minority varieties due to its diversity and territorial extension, offering the local viticulture the possibility of diversifying its wine production. The molecular identification of 98 specimens from six areas with a winemaking tradition in Andalusia was carried out between the years 2020 and 2022. Thirteen microsatellite markers were used in this study, including the nine recommended by the OIV. 31 different genotypes were obtained, 20 of which corresponded to profiles of already described varieties (11 of them are of minority cultivation in Andalusia: 'Rojal Tinto', 'Beba', 'Zurieles', 'Rome', 'Hebén', 'Mollar Cano', 'Listán Prieto', 'Listán del Condado', 'Jarrosuelto', 'Negra Dorada' and 'Mantúo de Pilas'), while the other 11 profiles did not match with previously identified varieties. This profiles were integrated into the database of the IFAPA “Rancho de la Merced” Germplasm Bank. The eco-geographical groups of the new identified genotypes were determined through analysis of genetic diversity. The presence of the Grapevine Fanleaf Virus, the Grapevine Fleck Virus and the Grapevine Leafroll associated Viruses was also analyzed using an ELISA test due to the necessity of having certifiable clones of the new varieties for their potential interest in being authorized for its cultivation in Spain.