Salvador Soler

Evolutionary analysis of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) causes severe economic losses in many crops worldwide and often overcomes resistant cultivars used for disease control. Comparison of nucleotide and amino acid sequences suggested that tomato resistance conferred by the gene Sw-5 can be overcome by the amino acid substitution C to Y at position 118 (C118Y) or T120N in the TSWV movement protein, NSm. Phylogenetic analysis revealed that substitution C118Y has occurred independently three times in the studied isolates by convergent evolution, whereas the substitution T120N was a unique event. Analysis of rates of non-synonymous and synonymous changes at individual codons showed that substitution C118Y was positively selected.

Comparison of the complete sequences of three different isolates of Pepino mosaic virus: Size variability of the TGBp3 protein between tomato and L. peruvianum isolates

Summary.The complete nucleotide sequence of the genomes of two Spanish isolates (LE-2000 and LE-2002) from tomato and one Peruvian isolate (LP-2001) from Lycopersicon peruvianum of the Pepino mosaic virus (PepMV) were determined. The tomato isolates share identities higher than 99%, while the genome of LP-2001 had mean nucleotide identities of 95.6% to 96.0% with tomato isolates. The predicted amino acid sequences showed similarities ranging between 95.2% and 100% with TGBp3 and TGBp2 and CP proteins, respectively. In LP-2001 two main differences were found with respect to the tomato isolates; (i) the 5′ untranslated region (UTR) was 2 nt shorter by deletion at position 12–13 and it had some polymorphims at the putative promoter sequence reported for PepMV tomato isolates and other potexviruses, which could be functionally significant for RNA replication, and (ii) the TGBp3 protein had two extra amino acids in the C-terminal region.

Effect of Temperature Regime and Growth Stage Interaction on Pattern of Virus Presence in TSWV-Resistant Accessions of Capsicum chinense

We studied the resistance to tomato spotted wilt virus in plant introduction accession (PI)-151225 and PI-159236 under Mediterranean climatic conditions. Two temperature regimes were utilized, corresponding to early and late cultivation cycles. Inoculations were made at 2- and 4-leaf stages to determine the effect of early infection. The existence of interaction between temperature regime and developmental stage was also studied. When plants of both PIs were maintained at 30/18°C (day/night), all plants at both growth stages when inoculated developed systemic infection. At 25/18°C, only those plants inoculated at 2-leaf stage became systematically infected; however, those inoculated at the 4-leaf stage behaved as resistant. Thus, there was an interaction between temperature regime and growth stage. There is potential for using this type of resistance in areas with mild climates, providing seedling infections are avoided.

Characterization of composition traits related to organoleptic and functional quality for the differentiation, selection and enhancement of local varieties of tomato from different cultivar groups.

Tomato (Solanum lycopersicum) local varieties are having an increasing demand. We characterized 69 local tomato accessions from eight cultivar groups for proximate composition traits, major sugars, acids and antioxidants. A large diversity was found, with differences among accessions of almost tenfold for lycopene. Significant differences were found among cultivar group means for most traits. The Cherry and Penjar groups generally presented higher dry matter, soluble solids content, titratable acidity, taste index, β-carotene, ascorbic acid, total phenolics, and antioxidant activity that the other groups. Wide ranges of variation were found within each cultivar group. Positive correlations were found between proximate traits related to taste and antioxidants. The multivariate principal components analysis confirms the distinct profile of the Cherry and Penjar groups and the large variation within groups. The results will be useful for the differentiation, enhancement and selection of local tomato varieties with improved organoleptic properties and functional quality.

Movement and distribution of tomato spotted wilt virus in resistant and susceptible accessions of Capsicum spp.

Resistant Capsicum chinense accessions PI-152225 and PI-159236 and the susceptible cultivar ‘Negral’ of Capsicum annuum were used in three experiments to characterize the resistance to tomato spotted wilt virus (TSWV) in these materials. In the first experiment viral movement in the whole plant was studied at two growth stages (2- and 4-leaf stage). In the second experiment the movement within the inoculated leaf was analysed at three growth stages (2-, 3-, and 4-leaf stage). Two techniques were used in this assay: double antibody sandwich – enzyme-linked immunosorbent assay (DAS–ELISA) and direct tissue blotting (DTB). In the latter experiment viral accumulation in different types of samples was evaluated by DAS–ELISA. The DTB technique showed that viral movement within the inoculated leaf is restricted in the resistant plants. The inoculated area was not totally infected in resistant accessions and slower viral movement within the inoculated area was observed. Detection of weak ELISA positives in inocul...

Detection, discrimination and absolute quantitation of Tomato spotted wilt virus isolates using real time RT-PCR with TaqMan(®)MGB probes.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan(®)MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidentalis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

Simultaneous detection of six RNA plant viruses affecting tomato crops using a single digoxigenin-labelled polyprobe

A polyprobe for the simultaneous detection by non-isotopic molecular hybridisation has been developed to detect any of the following six viruses causing important economic losses in tomato crops: Tomato spotted wilt virus, Tomato mosaic virus, Pepino mosaic virus, Cucumber mosaic virus, Potato Y virus and Parietaria mottle virus. The polyprobe detected all six viruses with similar sensitivity to that obtained using individual riboprobes. In addition, we evaluated the possible use of the tissue-printing as a sample preparation technique applied to routine diagnosis of tomato plants with the polyprobe.

Introgressiomics: a new approach for using crop wild relatives in breeding for adaptation to climate change

The need to boost agricultural production in the coming decades in a climate change scenario requires new approaches for the development of new crop varieties that are more resilient and more efficient in the use of resources. Crop wild relatives (CWRs) are a source of variation for many traits of interest in breeding, in particular tolerance to abiotic and biotic stresses. However, their potential in plant breeding has largely remained unexploited. CWRs can make an effective contribution to broadening the genetic base of crops and to introgressing traits of interest, but their direct use by breeders in breeding programs is usually not feasible due to the presence of undesirable traits in CWRs (linkage drag) and frequent breeding barriers with the crop. Here we call for a new approach, which we tentatively call ‘introgressiomics’, which consists of mass scale development of plant materials and populations with introgressions from CWRs into the genetic background of crops. Introgressiomics is a form of pre-emptive breeding and can be focused, when looking for specific phenotypes, or un-focused, when it is aimed at creating highly diverse introgressed populations. Exploring germplasm collections and identifying adequate species and accessions from different genepools encompassing a high diversity, using different strategies like the creation of germplasm diversity sets, Focused identification of germplasm strategy (FIGS) or gap analysis, is a first step in introgressiomics. Interspecific hybridization and backcrossing is often a major barrier for introgressiomics, but a number of techniques can be used to potentially overcome these and produce introgression populations. The generation of chromosome substitution lines (CSLs), introgression lines (ILs), or multi-parent advanced inter-cross (MAGIC) populations by means of marker-assisted selection allows not only the genetic analysis of traits present in CWRs, but also developing genetically characterized elite materials that can be easily incorporated in breeding programs. Genomic tools, in particular high-throughput molecular markers, facilitate the characterization and development of introgressiomics populations, while new plant breeding techniques (NPBTs) can enhance the introgression and use of genes from CWRs in the genetic background of crops. An efficient use of introgressiomics populations requires moving the materials into breeding pipelines. In this respect public–private partnerships (PPPs) can contribute to an increased use of introgressed materials by breeders. We hope that the introgressiomics approach will contribute to the development of a new generation of cultivars with dramatically improved yield and performance that may allow coping with the environmental changes caused by climate change while at the same time contributing to a more efficient and sustainable agriculture.

Complete nucleotide sequence of a Spanish isolate of Parietaria mottle virus infecting tomato.

The genome of a Spanish isolate of Parietaria mottle virus (PMoV) obtained from tomato (strain PMoV-T) was completely sequenced. Protein motifs conserved for RNA viruses were identified: the p1 protein contained a metyltransferase domain in its N-terminal half and a triphosphatase/helicase domain in its C-terminal half, the p2 protein contained a RNA polymerase domain; the 3a protein contained a RNA-binding domain with α-helix and β-sheet secondary structures. In addition, stem-loop structures with potential capacity of protein interactions were predicted on the untranslated terminal regions. Comparison with the other sequenced PMoV isolate showed nucleotide identities of 93, 90, and 93% for genomic RNAs 1, 2 and 3, respectively, and amino acid identities ranging from 88 to 97% for the different proteins. A cytosine deletion was detected at position 1,366 of RNA 3, involving a start codon for the coat protein (CP) gene different from the other PMoV isolate, resulting in a CP 16 amino acids shorter. Comparison of synonymous and nonsynonymous mutations revealed different selective constraints along the genome.

Tomato Mosaic Tobamovirus, Causal Agent of a Severe Disease of Pepino (Solanum muricatum)

Pepino (Solanum muricatum Aiton), a vegetatively propagated herbaceous crop from the Andes, is esteemed for its edible, juicy, and fragrant fruits. Its cultivation as a crop for diversification is increasing in many frost-free areas throughout the world (2). In 1994, a severe viruslike disease, previously undescribed, was observed on pepino plants in Valencia, Spain. The disease has continued to cause economic losses in pepino plantings in subsequent years. Symptoms, which are exacerbated at temperatures above 30°C, include dark and light green mosaic predominantly in young leaves, leaf puckering and distortion, short internodes, fruit deformation, delay in ripening, and yield reduction. Samples from affected plants were analyzed by enzyme-linked immunosorbent assay (ELISA). All samples displaying viruslike symptoms reacted positively with antiserum made against tomato mosaic tobamovirus (ToMV) but not with antisera made against alfalfa mosaic alfamovirus, cucumber mosaic cucumovirus, potato Y potyvirus, tobacco mosaic tobamovirus, tomato spotted wilt tospovirus, or tomato yellow leaf curl bigeminivirus. A leaf extract from diseased plants was heated at 72°C for 10 min. This treatment inactivates most plant viruses but does not eliminate infectivity of ToMV (1). Inoculation of a collection of pepino clones resulted in the development of symptoms in most clones. Symptomatic clones were also ELISA-positive for ToMV. A few clones showed a hypersensitive reaction, which consisted of the development of necrotic lesions in the inoculated area. New growth on these clones was asymptomatic and ELISA-negative for ToMV. These results clearly point to a causal relationship between ToMV infection and the observed disease. The initial source of the infection should be eliminated from commercial plantings, as ToMV is easily transmitted when the pepino plants are trellised and pruned. Special care must also be taken to ensure that mother plants from which cuttings are taken are free from this virus. In addition, ToMV is usually found in meristematic tissues, even after thermotherapy and chemotherapy treatments are applied, making the regeneration of virus-free plants from infected clones by meristem tip culture difficult. Therefore, it seems that the best strategy against this disease is the development of resistant cultivars. References: (1) H. Laterrot. Ann. Amélior. Plant. 23:287, 1973; (2) J. Prohens et al. Econ. Bot. 50:355, 1996.