RESUMO
The Ty-1 locus confers tolerance to monopartite and bipartite Begomovirus spp. (genus Begomovirus, family Geminiviridae) and this phenotype is improved in homozygous tomato lines. However, the gene Mi (Meloidogyne spp. resistance) is in repulsion phase linkage with Ty-1, which hampers the large-scale development of multiresistant inbred lines. Seventy-one Solanum (section Lycopersicon) accessions were whitefly inoculated with the bipartite Begomovirus sp. Tomato rugose mosaic virus (ToRMV) and simultaneously infested with a mixture of Meloidogyne incognita and M. javanica under greenhouse conditions in Brazil. Accessions were then transplanted into a nematode-infested field with natural ToRMV infection. A severity index was used to evaluate ToRMV reaction. Nematode evaluation was done by counting the number of galls per root system. Seventeen accessions with Meloidogyne spp. and ToRMV resistance were selected and evaluated in Spain against three monopartite Begomovirus spp. associated with the tomato yellow leaf curl virus disease, using infectious clones. Systemic infection was monitored by DNA hybridization. Five S. peruvianum accessions (PI-306811, PI-365951, LA-1609, LA-2553, and CNPH-1194) displayed nematode and broad-spectrum resistance to all Begomovirus spp. tested in both continents. From the breeding standpoint, accessions combining resistance to Meloidogyne spp. and to bipartite and monopartite Begomovirus spp. would be useful for the development of elite lines expressing all traits in homozygous condition.
RESUMO
Torrao or torrado is an emerging disease that is causing serious economic losses in tomato crops of southeastern Spain. The causal agent has been shown to be a new picorna-like plant virus, tentatively named Tomato torrado virus (ToTV) (4). By using trap tomato plants in a greenhouse affected by torrado located in the Murcia Region of Spain, we obtained a ToTV isolate (ToTV-CE) that we have biologically and molecularly characterized. Subtracted cDNA libraries (1) and expressed sequence tags sequencing were used to determine the partial nucleotide sequence of ToTV-CE. We covered ≈53% of the virus genome (GenBank Accession Nos. EU476181 and EU476182) and found that ToTV-CE RNAs 1 and 2 had a high nucleotide similarity (98 and 99%, respectively) with the ToTV published sequences (2,4). ToTV-CE sequences also showed a 70% nt similarity with those of Tomato apex necrosis virus, a newly identified virus in tomato crops of the Culiacan area (Sinaloa, Mexico) (3). To characterize the host range of ToTV-CE, 6 to 10 plants belonging to 14 species were mechanically inoculated with extracts from ToTV-CE-infected Nicotiana benthamiana plants. The presence of ToTV in these plants was analyzed at 3 and 6 weeks postinoculation (PI) by molecular hybridization in dot-blots. The determined host range was in agreement with that described earlier (2,4), but additional hosts and nonhosts were identified. Thus, the virus did not infect melon (Cucumis melo var. cantaloupe), cucumber (C. sativus cv. Marketmore), squash (Cucurbita pepo cv. Negro Belleza), Chenopodium album ssp. Amaranticolor, or Chenopodium quinoa. The virus infected systemically N. benthamiana, N. glutinosa, N. rustica, tobacco (N. tabacum cvs. Xanthi nc and Samsun), Physalis floridana, pepper (Capsicum annuum cv. Italian Long Sweet), tomato (Solanum lycopersicum cv. Boludo), and eggplant (S. melongena cv. Black Beauty). Pepper plants displayed severe symptoms of infection consisting of marked mosaics and stunting (but no necrosis), but eggplant remained asymptomatic for up to 6 weeks PI. A simple assay was devised to analyze whether ToTV can be transmitted by whiteflies. ToTV-CE-infected tomato plants were placed together with three to eight healthy tomato seedlings inside insect-proof glass boxes. Adult Bemisia tabaci (100 to 800 individuals in three replicates) or Trialeurodes vaporariorum (100 individuals in one replicate) were released into each box. For both treatments, symptoms typically induced by ToTV appeared in one to seven tomato seedlings by 1 week after the release of the whiteflies. ToTV infection was confirmed by molecular hybridization in tissue prints of petiole cross sections at 10 days PI. These data are in agreement with those reported by Pospieszny et al. (2) and strongly suggest that both B. tabaci and T. vaporariorum can transmit ToTV. References: (1) L. Diachenko et al. Proc. Natl. Acad. Sci. USA 93:6025, 1996. (2) H. Pospieszny et al. Plant Dis. 91:1364, 2007 (3) M. Turina et al. Plant Dis. 91:932, 2007. (4) M. Verbeek et al. Arch. Virol. 152:881, 2007.
RESUMO
The genetic structure of the population of Watermelon mosaic virus (WMV) in Spain was analysed by the biological and molecular characterisation of isolates sampled from its main host plant, melon. The population was a highly homogeneous one, built of a single pathotype, and comprising isolates closely related genetically. There was indication of temporal replacement of genotypes, but not of spatial structure of the population. Analyses of nucleotide sequences in three genomic regions, that is, in the cistrons for the P1, cylindrical inclusion (CI) and capsid (CP) proteins, showed lower similar values of nucleotide diversity for the P1 than for the CI or CP cistrons. The CI protein and the CP were under tighter evolutionary constraints than the P1 protein. Also, for the CI and CP cistrons, but not for the P1 cistron, two groups of sequences, defining two genetic strains, were apparent. Thus, different genomic regions of WMV show different evolutionary dynamics. Interestingly, for the CI and CP cistrons, sequences were clustered into two regions of the sequence space, defining the two strains above, and no intermediary sequences were identified. Recombinant isolates were found, accounting for at least 7% of the population. These recombinants presented two interesting features: (i) crossover points were detected between the analysed regions in the CI and CP cistrons, but not between those in the P1 and CI cistrons, (ii) crossover points were not observed within the analysed coding regions for the P1, CI or CP proteins. This indicates strong selection against isolates with recombinant proteins, even when originated from closely related strains. Hence, data indicate that genotypes of WMV, generated by mutation or recombination, outside of acceptable, discrete, regions in the evolutionary space, are eliminated from the virus population by negative selection.
