Alberto Yuste

Real-time multiplex RT-PCR for the simultaneous detection of the five main grapevine viruses.

A real-time multiplex RT-PCR has been developed for the simultaneous detection and identification of the major RNA viruses that infect grapevines (Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3 and Grapevine fleck virus). Serial dilutions of infected plant extracts were tested using the new method, and the results were compared with those obtained using a commercially available ELISA and real-time singleplex RT-PCR. The two real-time RT-PCR versions detected up to the same level of dilution and were at least 10,000 times more sensitive than the ELISA. In addition, 158 grapevine plants collected in a survey of the Protected Designation of Origin in Alicante, Spain were compared using the three methods. The results of the molecular methods were very similar, with only four discordant results, and both were able to detect many more infected plants than the ELISA. The high prevalence of Grapevine fleck virus, Grapevine leafroll-associated virus 3 and Grapevine fanleaf virus suggests that the main pathways of viral introduction are infected plant material that has escaped controls and/or uncontrolled traffic of propagating plant material. Real-time multiplex RT-PCR could be used to facilitate a better control of grapevine viruses.

Ingenane and lathyrane diterpenes from the latex of Euphorbia canariensis

The latex of Euphorbia canariensis yielded, in addition to five known ingenol esters, the ingenane derivatives ingenol 3-angelate 5,20-diacetate and 5-deoxyingenol 3-angelate 20-acetate, and the lathyrane derivatives 2,3-diepiingol 7,12-diacetate 8-benzoate, 2,3-diepiingol 7,12-diacetate 8-isobutyrate and 2-epiingol 3,7,12-triacetate 8-benzoate. The structures were established with the aid of spectroscopic methods, mainly NMR, and molecular mechanics calculations. They were also supported by the results of some chemical transformations.

Sesquiterpene lactones fromArtemisia barrelieri

Abstract Extraction of aerial parts of Artemisia barrelieri and chromatographic separation yielded a new germacranolide, two new monocyclic sesquiterpenes and a novel germacranolide dimer, together with several known compounds.

High prevalence of viruses in table grape from Spain detected by real-time RT-PCR

Table grapes from one of the most important growing area in Spain (Vinalopó, Alicante) protected by the Designation of Origin “Vinalopó bagged table grape”, were surveyed and analysed to determine the prevalence of the five viruses included in the Spanish certification program: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Grapevine leafroll associated virus-1 (GLRaV-1) and Grapevine leafroll associated virus-3 (GLRaV-3). Ninety five sampling points were selected and the position of grapevine plants georeferenced. Samples were collected in two different vegetative periods and analyses were performed by ELISA and real-time RT-PCR. Purified RNA and immobilized viral targets from plant extracts on nylon membranes were used in parallel assays as templates for PCR assays. In order to analyse these five viral species by real-time RT-PCR, new specific primers and TaqMan probes were designed for detection of ArMV and GFkV. Real time RT-PCR from purified RNA was more sensitive than spot version and ELISA tests. The most prevalent virus was GFLV (95.8%) followed by GLRaV-3 (94.7%), GLRaV-1 (66.3%) and GFkV (65.3%). ArMV was not detected in any sample. The high level of viral infections and the presence of mixed infections suggest that initial infected plant material and uncontrolled traffic of propagation material have played an important role in the spread of viruses.

Jatrophane derivatives and a rearranged jatrophane from Euphorbia terracina

Abstract The aerial parts of Euphorbia terracina yielded 11 new jatrophane derivatives as well as a further diterpene displaying the novel 1(15 → 14) abeo -jatrophane framework.

Sesquiterpene lactones and dihydroflavonols from Andryala and Urospermum species

Abstract The aerial parts of two Andryala species yielded several guaianolides and guaianolide glycosides, three of them being new. The aerial parts of Urospermum dalechampii yielded several known germacranolides and the new dihydroflavonol 3- O -methyltaxifolin.

Terracinolides from Euphorbia terracina

Abstract The aerial parts of Euphorbia terracina yielded five new bishomoditerpene lactones, named terracinolides C-G, which display the novel C 22 17-ethyljatrophane framework.

Comparative Analysis of the Vascular Actions of Diterpenes Isolated from Euphorbia canariensis

We have analysed the effects of 2,3‐diepiingol 7,12‐diacetate‐8‐isobutyrate (compound 1), ingenol‐3‐angelate‐17‐benzoate (compound 2), ingenol‐3‐angelate‐17‐benzoate‐20‐acetate (compound 3) and 3,5,7,8,9,15‐hexahydroxyjatropha‐6(17),11‐dien‐14‐one‐5,8‐bis(2‐methylbutyrate)‐7‐(2‐methylpropionate) (compound 4), four diterpenes isolated from E. canariensis, on the isometric tension developed by isolated rabbit basilar and carotid arteries.

A novel germacranolide-aminoacid adduct dimer from centaurea aspera

Abstract The novel germacronolide-valine adduct dimer 1 has been isolated from aerial parts of Centaurea aspera var. aspera . Its structure has been determined by spectroscopic methods.

Analysis of rabbit vascular responses to DBI, an ingol derivative isolated from Euphorbia canariensis.

We have analysed the effects of 7,12‐O‐diacetyl‐8‐O‐benzoil‐2,3‐diepiingol (DBI), an ingol derivative isolated from E. canariensis, on isometric tension developed by isolated rabbit basilar and carotid arteries.