In situ hybridization for the localization of two pepino mosaic virus isolates in mixed infections.

In situ hybridization (ISH) is an informative and relatively accessible technique for the localization of viral genomes in plant tissue and cells. However, simultaneous visualization of related plant viruses in mixed infections may be limited by the nucleotide similarity in the genomes and the single chromogenic detection over the same sample preparation. To address this issue, we used two Pepino mosaic virus isolates and performed ISH over consecutive serial cross-sections of paraffin-embedded leaf samples of single and mixed infected Nicotiana benthamiana plants. Moreover, the probe design was optimized to reduce cross-hybridisation, and co-localization was based on the overlapping of consecutive cross-sections from mixed infected leaves; thus, our results showed that both Pepino mosaic virus isolates co-localized in the same leaf tissue. In turn, both isolates were localized in the cytoplasm of the same cells. These results provide valuable information for studying mixed infections in plants by using a simple ISH procedure that is accessible to any pathology laboratory.

Tomato torrado virus is Transmitted by Bemisia tabaci and Infects Pepper and Eggplant in Addition to Tomato.

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.

A century of tobamovirus evolution in an Australian population of Nicotiana glauca.

The evolution over the past century of two tobamoviruses infecting populations of the immigrant plant Nicotiana glauca in New South Wales (NSW), Australia, has been studied. This plant species probably entered Australia in the 1870s. Isolates of the viruses were obtained from N. glauca specimens deposited in the NSW Herbarium between 1899 and 1972, and others were obtained from living plants in 1985 and 1993. It was found that the NSW N. glauca population was infected with tobacco mosaic tobamovirus (TMV) and tobacco mild green mosaic tobamovirus (TMGMV) before 1950 but only with TMGMV after that date. Half the pre-1950 infections were mixtures of the two viruses, and one was a recombinant. Remarkably, sequence analyses showed no increase in the genetic diversity among the TMGMV isolates over the period. However, for TMV, the genetic diversity of synonymous (but not of nonsynonymous) differences between isolates varied and was correlated with their time of isolation. TMV accumulated to smaller concentrations than TMGMV in N. glauca plants, and in mixed experimental infections, the accumulation of TMV, but not of TMGMV, was around 1/10 that in single infections. However, no evidence was found of isolate-specific interaction between the viruses. We conclude that although TMV may have colonized N. glauca in NSW earlier or faster than TMGMV, the latter virus caused a decrease of the TMV population below a threshold at which deleterious mutations were eliminated. This phenomenon, called Muller's ratchet, or a "mutational meltdown," probably caused the disappearance of TMV from the niche.

Genetic diversity in tobacco mild green mosaic tobamovirus infecting the wild plant Nicotiana glauca.

The variability and genetic structure of tobacco mild green mosaic tobamovirus (TMGMV) was analyzed in a collection of isolates from Nicotiana glauca plants from Australia, California, Spain, and the east Mediterranean Basin. Ribonuclease protection was assayed on 53 isolates with six probes representing 85% of the TMGMV genome. Results showed that conserved domains in the TMGMV genome were different for each geographic population. The nucleotide sequence of two regions of the TMGMV genome was determined for 33 isolates. Nucleotide diversity values were smaller than those reported for other RNA genomes. For each population, genetic diversity was not related to the size of the area from which the isolates were collected, and was particularly small for Australia and Spain. Diversity values indicated population differentiation, which was largest between the less diverse Spanish and Australian populations. Nucleotide diversity values for nonsynonymous vs synonymous positions indicate moderate negative selection, except for the Spanish population for which it gave evidence of positive selection. On the whole, our data indicate that the low genetic diversity for this plant virus may be due to different factors such as positive and negative selection or the recent colonization of new areas or host plants. The intensity of these factors appears to be different for the various TMGMV geographical populations.

Experimental evaluation of the ribonuclease protection assay method for the assessment of genetic heterogeneity in populations of RNA viruses.

The ribonuclease (RNase) protection assay (RPA) was evaluated as a method to estimate genetic distances among sequence variants of RNA viruses. The patterns of fragments generated, under different RPA conditions, by three sets of RNA sequence variants of known nucleotide sequence, were analyzed. Both the effectiveness of cleavage (i.e. the probability of cleavage in a certain heteroduplex) and its degree (i.e. in all the molecules in the assay or in a part of them) varied largely according to the nature of the mismatch. Probability and degree of cleavage were also dependent on distant sequence context effects. No correlation could be established between context and cleavage, so that the pattern of fragments in RPA cannot be unequivocally predicted from sequence information. Accordingly, nucleotide sequence differences between two sequence variants cannot be directly derived from RPA data. For all three sequence sets linear relationships were found between the number of non-shared fragments in the RPAs of two variants and their nucleotide sequence differences. Nevertheless, both linearity and the linear regression parameters varied largely according to the sequence set and according to RPA conditions, in a non-predictable way. Thus, under experimental conditions, RPA may not be as appropriate a method to estimate genetic distances between RNA sequences as simulation under an ideal model suggested. Possible ways to diminish the gap between the ideal model and the experimental procedure are proposed.

Increase in the relative fitness of a plant virus RNA associated with its recombinant nature.

A recombination event at the 3' end of RNA 3 in a pseudorecombinant virus (C1C2T3) having RNAs 1 and 2 from TrK7-cucumber mosaic virus (CMV) and RNA 3 from P-tomato aspermy virus (TAV) was detected by ribonuclease protection assay (RPA). Sequence analysis of the 3' end of RNA 3 of C1C2T3 and of its parental CMV and TAV strains showed that RNA 3 of C1C2T3 had a hybrid nature in which most of the 3' noncoding region (3' ncr), including the whole 3' terminal tRNA-like structure, was derived from CMV RNA 2 by recombination. Recombination may have occurred in two steps. The first one would have caused the duplication of a large region 5' to the 3' end tRNA-like structure, derived from TAV and from CMV. The second one would have eliminated the TAV-derived sequences in this duplicated region. Competition experiments in tobacco plants showed that in a context of RNA 1 + 2 from CMV, RNA 3 from TAV is outcompleted by RNA 3 of CMV, but the recombinant TAV RNA 3 with the 3' end of CMV outcompetes both TAV and CMV RNA 3. This shows an increase in relative fitness associated with the recombinant nature of RNA 3. Our results document the potential importance of RNA-RNA recombination in the determination of the genetic structure of RNA viral populations.