Renato O. Resende

Synergistic Interaction Between Tomato chlorosis virus and Tomato spotted wilt virus Results in Breakdown of Resistance in Tomato

ABSTRACT Multiple viral infections frequently are found in single plants of cultivated and wild hosts in nature, with unpredictable pathological consequences. Synergistic reactions were observed in mixed infections in tomato plants doubly infected with the positive-sense and phloem-limited single-stranded RNA (ssRNA) crinivirus Tomato chlorosis virus (ToCV) and the negative-sense ssRNA tospovirus Tomato spotted wilt virus (TSWV). Synergism in a tomato cultivar susceptible to both viruses resulted in a rapid death of plants. A pronounced enhancement of ToCV accumulation mediated by TSWV co-infection was observed with no evident egress of ToCV from phloem tissues. No consistent alteration of TSWV accumulation was detected. More remarkable was the synergism observed in tomato cultivars which carry the Sw-5 resistance gene, which are resistant to TSWV. Pre-infection with ToCV resulted in susceptibility to TSWV, whereas co-inoculations did not. This suggested that a threshold level or a time lapse is needed for ToCV to interfere or downregulate the defense response in the TSWV-resistant plants.

The identification of the vector species of iris yellow spot tospovirus occurring on onion in Brazil.

In Brazil, tospoviruses have been reported in several horticultural and ornamental plants. In the northeast region of Brazil, a tospovirus has emerged as a devastating virus on onion cultures. Based on serology and the sequence of nucleocapsid (N) protein gene, this pathogen was identified as a strain of iris yellow spot tospovirus (IYSV) (1). This virus was first identified on iris and leek in The Netherlands and later on onion in Israel. For an effective integrated management of tospoviruses in Brazil, identification of IYSV vector is essential. Three thrips species, Thrips tabaci, Frankliniella schultzei, and F. occidentalis, that are major vegetable and floral crop pests in the Federal District, Brazil, were tested for their ability to transmit the virus by leaf disk assay (2). All thrips, up to 8 h old, were given an acquisition access period of 48 h at 25°C on IYSV-infected Nicotiana benthamiana plants in Tashiro-cages. Thrips were then reared on uninfected Datura stramonium detached leaves until the adult stage. These adults were transferred individually to microcentrifuge tubes containing an N. benthamiana leaf disk and were incubated for 48 h for virus inoculation. The leaf disks were then incubated 4 more days to allow development of the virus infection, and the presence of virus was evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) with polyclonal antibodies against N protein of IYSV. Adult thrips were also used for direct inoculation to N. benthamiana plants, three thrips per plant. By the leaf disk assay, 45.8% (22 out of 48) of T. tabaci transmitted the virus, but F. schultzei (n = 48) and F. occidentalis (n = 32) did not transmit it. All plants (4 out of 4) directly inoculated by T. tabaci showed symptoms and infection by Dot-ELISA, while no plants inoculated with F. schultzei (n = 5) and F. occidentalis (n = 3) were positive, either by symptom observation or by Dot-ELISA. Only T. tabaci showed potential for a high capacity to transmit the IYSV onion isolate. In the field, considering the host preference of thrips, T. tabaci was considered the most important vector species of IYSV on onion. References: (1) L. Pozzer et al. Plant Dis. (In press.) (2) I. Wijkamp and D. Peters. Phytopathology 83:986, 1993.

Sequence diversity of NSM movement protein of tospoviruses

Summary. In order to determine the diversity of the movement protein (NSM) among tospoviruses, the NSM genes of five distinct tospovirus species occurring in Brazil (Tomato chlorotic spot virus, Groundnut ring spot virus, Chrysanthemum stem necrosis virus, Zucchini lethal chlorosis virus and Iris yellow spot virus) were cloned, sequenced and compared with NSM sequences of other available tospoviruses. The ‘D-motif’, a conserved region present in the majority of ‘30K superfamily’ virus movement proteins, is present in all NSM amino acid sequences available. In addition to the ‘D-motif’, a conserved phospholipase A2 motif was found. The NSM amino acid sequence comparisons among tospovirus species revealed several conserved regions located in the internal part of the protein and diverse domains mainly located in the amino-terminus. Prediction of secondary structure showed similar patterns among all NSM proteins analyzed. Considering the geographical prevalence and phylogenetic analysis of N and NSM proteins, tospoviruses were tentatively clustered in ‘American’ and ‘Eurasian’ groups. Both phylogenetic trees may reflect the natural evolution of tospovirus species within distinct ecological niches. The sequence information obtained in this work would facilitate functional analysis of NSM during the tospovirus infection process.

Characterization of Bean Necrotic Mosaic Virus: A Member of a Novel Evolutionary Lineage within the Genus Tospovirus

Background Tospoviruses (Genus Tospovirus, Family Bunyaviridae) are phytopathogens responsible for significant worldwide crop losses. They have a tripartite negative and ambisense RNA genome segments, termed S (Small), M (Medium) and L (Large) RNA. The vector-transmission is mediated by thrips in a circulative-propagative manner. For new tospovirus species acceptance, several analyses are needed, e.g., the determination of the viral protein sequences for enlightenment of their evolutionary history. Methodology/Principal Findings Biological (host range and symptomatology), serological, and molecular (S and M RNA sequencing and evolutionary studies) experiments were performed to characterize and differentiate a new tospovirus species, Bean necrotic mosaic virus (BeNMV), which naturally infects common beans in Brazil. Based upon the results, BeNMV can be classified as a novel species and, together with Soybean vein necrosis-associated virus (SVNaV), they represent members of a new evolutionary lineage within the genus Tospovirus. Conclusion/Significances Taken together, these evidences suggest that two divergent lineages of tospoviruses are circulating in the American continent and, based on the main clades diversity (American and Eurasian lineages), new tospovirus species related to the BeNMV-SVNaV clade remain to be discovered. This possible greater diversity of tospoviruses may be reflected in a higher number of crops as natural hosts, increasing the economic impact on agriculture. This idea also is supported since BeNMV and SVNaV were discovered naturally infecting atypical hosts (common bean and soybean, respectively), indicating, in this case, a preference for leguminous species. Further studies, for instance a survey focusing on crops, specifically of leguminous plants, may reveal a greater tospovirus diversity not only in the Americas (where both viruses were reported), but throughout the world.

Pepper yellow mosaic virus, a new potyvirus in sweetpepper, Capsicum annuum

Summary. A potyvirus was found causing yellow mosaic and veinal banding in sweetpepper in Central and Southeast Brazil. The sequence analysis of the 3′ terminal region of the viral RNA revealed a coat protein of 278 amino acids, followed by 275 nucleotides in the 3′-untranslated region preceding a polyadenylated tail. The virus shared 77.4% coat protein amino acid identity with Pepper severe mosaic virus, the closest Potyvirus species. The 3′-untranslated region was highly divergent from other potyviruses. Based on these results, the virus found in sweetpepper plants could be considered as a new potyvirus. The name Pepper yellow mosaic virus (PepYMV) is suggested.

Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virusfree garlic was evaluated.

First Report of Natural Occurrence of Zucchini Lethal Chlorosis Tospovirus on Cucumber and Chrysanthemum Stem Necrosis Tospovirus on Tomato in Brazil

During a field survey in 1994, five cucumber (Cucumis sativus) cv. Hokushin plants showing symptom of yellowing, mottling, and vein banding on the leaves were collected from a commercial field of the Federal District. By electron microscopy, quasi-spherical particles with double membrane, typical tospovirus-like particles were found in the infected leaf material. All samples strongly reacted with antibody of zucchini lethal chlorosis tospovirus (ZLCV), but not with antibodies of other to-spoviruses reported in Brazil (1): tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), groundnut ringspot virus (GRSV), chrysanthemum stem necrosis virus (CSNV), or iris yellow spot virusonion isolate (IYSV-BR). The virus was identified as ZLCV, which was first isolated in 1994 from zucchini (Cucurbita pepo) in São Paulo State, Brazil. Tomato (Lycopersicon esculentum) plants showing stem necrosis and necrotic spots and rings on the leaves were collected in Viçosa, Minas Gerais State. By electron microscopy, molecular studies, and enzyme-linked immunosorbent assay with antibodies of the six tospoviruses occurring in Brazil, the virus was identified as CSNV. This virus was first reported in 1995 on a Chrysanthemum sp. in São Paulo State and recently reported in the Netherlands from Dendranthema indicum. This is the first report of the natural occurrence of ZLCV and CSNV on cucumber and tomato, respectively. Reference: (1) A. C. de Ávila et al. 1998. Pages 32-34 in: Int. Symp. on Tospoviruses and Thrips in Floral and Vegetable Crops, 4th.

The N protein of Tomato spotted wilt virus (TSWV) is associated with the induction of programmed cell death (PCD) in Capsicum chinense plants, a hypersensitive host to TSWV infection.

In sweet pepper, the Tsw gene, originally described in Capsicum chinense, has been widely used as an efficient gene for inducing a hypersensitivity response (HR) derived Tomato spotted wilt virus (TSWV) resistance. Since previously reported studies suggested that the TSWV-S RNA mutation(s) are associated with the breakdown of Tsw mediated TSWV resistance in peppers, the TSWV genes N (structural nucleocapsid protein) and NS(S) (non-structural silencing suppressor protein) were cloned into a Potato virus X (PVX)-based expression vector, and inoculated into the TSWV-resistant C. chinense genotype, PI 159236, to identify the Tsw-HR viral elicitor. Typical HR-like chlorotic and necrotic lesions followed by leaf abscission were observed only in C. chinense plants inoculated with the PVX-N construct. Cytopathological analyses of these plants identified fragmented genomic DNA, indicative of programmed cell death (PCD), in mesophyll cell nuclei surrounding PVX-N-induced necrotic lesions. The other constructs induced only PVX-like symptoms without HR-like lesions and there were no microscopic signs of PCD. The mechanism of TSWV N-gene HR induction is apparently species specific as the N gene of a related tospovirus, Tomato chlorotic spot virus, was not a HR elicitor and did not cause PCD in infected cells.

Genetic diversity and recombination analysis of sweepoviruses from Brazil

BackgroundMonopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect sweet potato (Ipomoea batatas) around the world are known as sweepoviruses. Because sweet potato plants are vegetatively propagated, the accumulation of viruses can become a major constraint for root production. Mixed infections of sweepovirus species and strains can lead to recombination, which may contribute to the generation of new recombinant sweepoviruses.ResultsThis study reports the full genome sequence of 34 sweepoviruses sampled from a sweet potato germplasm bank and commercial fields in Brazil. These sequences were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic diversity and patterns of genetic exchange in sweepoviruses isolated from Brazil, as well as to review the classification and nomenclature of sweepoviruses in accordance with the current guidelines proposed by the Geminiviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV). Co-infections and extensive recombination events were identified in Brazilian sweepoviruses. Analysis of the recombination breakpoints detected within the sweepovirus dataset revealed that most recombination events occurred in the intergenic region (IR) and in the middle of the C1 open reading frame (ORF).ConclusionsThe genetic diversity of sweepoviruses was considerably greater than previously described in Brazil. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of sweepovirus species and strains and provided valuable new information for understanding the diversity and evolution of sweepoviruses.

Development of a locus-specific, co-dominant SCAR marker for assisted-selection of the Sw-5 (Tospovirus resistance) gene cluster in a wide range of tomato accessions

The best levels of broad-spectrum Tospovirus resistance reported in tomatoes thus far are conferred by the Sw-5 locus. This locus contains at least five paralogues (denoted Sw-5a through Sw-5e), of which Sw-5b represents the actual resistance gene. Here we evaluated a panel of seven PCR primer pairs matching different sequences within a genomic region spanning the Sw-5a and Sw-5b gene cluster. Primer efficiency evaluation was done employing tomato isolines with and without the Sw-5 locus. One primer pair produced a single and co-dominant polymorphism between susceptible and resistant isolines. Sequence analysis of these amplicons indicated that they were specific for the Sw-5 locus and their differences were due to insertions/deletions. The polymorphic SCAR amplicon encompass a conserved sequence of the promoter region of the functional Sw-5b gene, being located in the position −31 from its open reading frame. This primer pair was also evaluated in field assays and with a collection of accessions known to be either susceptible or resistant to tospoviruses. An almost complete correlation was found between resistance under greenhouse/field conditions and the presence of the marker. Therefore, this primer pair is a very useful tool in marker-assisted selection systems in a large range of tomato accessions.