Detección y caracterización de virus epidemiológicamente relevantes en cultivos de tomate y cucurbitáceas

Résumé

Tomatoes and cucurbits are among the most important cultivated vegetables and Spain is one of its main world producers. Virosis pose a serious threat to these crops and can lead to significant economic losses worldwide. Given the difficulty in controlling viral diseases, it is essential to have adequate detection techniques that allow, not only to monitor the development of the disease, but also to adequately implement preventive and containment measures. The use of resistant varieties and the use of plant material free of viruses are among the main measures of protection against viruses. In this context the work of this thesis has been developed, which is structured in two chapters. The first chapter describes an epidemiological study about aggressive outbreaks attributable to tomato yellow leaf curl disease (TYLCD) in tomato plants carrying TYLCV resistance genes. This study was carried out during the years 2015 and 2016 in Murcia and during the year 2017 in Almería. Using Hight throughput sequencing (HTS) techniques (2015) and cloning and Sanger sequencing (2016 and 2017), a total of 28 TYLCV complete sequences were determined. The phylogenetic analyses carried out showed that all sequences belonged to TYLCV isolates of the Israel strain (TYLCV-IL, family: Geminiviridae, genus: Begomovirus). A first analysis made with the sequences of 2015 and 2016 showed the existence of two groups of isolates, one related to ancient isolates preexisting in the area, and another group, recently introduced, related to recombinant Moroccan isolates called TYLCV-IS76. An infectious clone generated from one of the sequences of this second group was used in an efficacy trial versus a TYLCV-IS76 clone, in which was observed that while IS76 predominated in mixed infection, in simple infection the Murcian isolate surpassed the recombinant. In the analysis of the sequences obtained in 2017, the presence of isolates of the TYLCV-IS76 type was identified for the first time, being the only evidence of this type of recombinant isolates outside Morocco. The second chapter of this thesis describes the development of the RT-qPCR technique combined with TaqMan probes for the detection of 3 cucurbit seed-borne viruses, cucumber green mottle mosaic virus (CGMMV) which belongs to Virgaviridae family, genus Tobamovirus, squash mosaic virus (SqMV), a member of the Secoviridae family, genus Comovirus, and melon necrotic spot virus (MNSV) which belongs to Tombusviridae family, genus Gammacarmovirus, and its comparison with the ELISA technique, which is the main technique used in certification analysis. With Taqman technique, the three viruses could be detected with great sensitivity and specificity, opening the possibility for multiplex analyses. The comparison with the ELISA technique, performed only with CGMMV and SqMV infected seeds, showed a higher sensitivity, both at the seed lot and at the detection limit levels, demonstrating its use suitability for seed analysis. Overall, the results of this thesis therein have allowed, on the one hand, to provide information on the current situation of TYLCV epidemics in Spain, and on the other hand, to design a rapid and sensitive method for the detection of 3 seed-borne viruses in cucurbits, opening the possibility of their implementation in future certification programs.