Tomato-infecting begomoviruses have been reported throughout Brazil since the introduction of the B biotype of Bemisia tabaci. Here, we report a large scale survey on the distribution and genetic diversity of tomato-infecting begomoviruses. Tomato samples with typical begomovirus symptoms were collected in seven different states, comprising the major tomato growing areas of the country. Viruses were detected by polymerase chain reaction (PCR) using universal primers for the genus Begomovirus. PCR-amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, at least seven previously undescribed species of begomoviruses were found. Four of the new viruses were found exclusively in the Southeastern states, two exclusively in the Northeastern states, and one was found in both regions. Sequence comparisons reveal strong evidence of recombination among the Brazilian begomoviruses. Together, the results indicate the existence of a high degree of pre-existing genetic diversity among tomato-infecting begomoviruses in Brazil and suggest that these viruses have emerged after being transferred from natural hosts to tomatoes, due to the introduction into Brazil of a novel polyfagous biotype of the whitefly vector.
Information on the distribution and prevalence of the economically destructive Begomovirus species and recombinant forms infecting fresh-market and processing tomato crops in Brazil is crucial in guiding breeding programs and also to understand the evolutionary mechanisms associated with the upsurge of so many species and quasi-species comprising this unique disease complex. An extensive survey was carried out over 3 years (between 2002 and 2004) aiming to study the diversity of begomoviruses in tomato plants, predominantly collected in central Brazil. Polymerase chain reaction (PCR) with degenerated primers was used to detect the begomoviruses in tomato leaf samples showing virus-like symptoms in commercial fields. Seven hundred and seventeen out of 2,295 samples were found to be PCR positive for a begomovirus infection. High quality sequences were obtained from a fragment encompassing the 5' region of the coat protein (CP) gene and a segment of the intergenic region for 295 isolates from distinct geographic regions. Comparison analyses with those available in public databases enabled preliminary classification of the isolates into four previously described and/or proposed species: Tomato severe rugose virus (61%), Tomato golden vein virus (29.8%), Tomato mottle leaf curl virus (7.1%), Tomato yellow vein streak virus (0.7%), and two putative new species (1.4% of isolates). Within the prevailing species, we noted a relatively low degree of diversity, possibly indicating the existence of recent population founder effects and/or recent selective sweeps.
During a survey conducted in several different regions of Brazil, two unique tospoviruses were isolated and characterized, one from chrysanthemum and the other from zucchini. The chrysanthemum virus displayed a broad host range, whereas the virus from zucchini was restricted mainly to the family Cucurbitaceae. Double-antibody sandwich-enzyme-linked immunosorbent assay and western immunoblot analyses demonstrated that both viruses were serologically distinct from all reported tospovirus species including the recently proposed peanut yellow spot virus and iris yellow spot virus (IYSV) species. The nucleotide sequences of the nucleocapsid (N) genes of both viruses contain 780 nucleotides encoding for deduced proteins of 260 amino acids. The N proteins of these two viruses displayed amino acid sequence similarities with the previously described tospovirus species ranging from 20 to 75%, but they were more closely related to each other (80%). Based on the biological and molecular features, these viruses are proposed as two new tospovirus species, designated chrysanthemum stem necrosis virus (CSNV) and zucchini lethal chlorosis virus (ZLCV). With the identification of CSNV and ZLCV, in addition to tomato spotted wilt virus, groundnut ring spot virus, tomato chlorotic spot virus, and IYSV, Brazil harbors the broadest spectrum of tospovirus species reported.
The nucleotide sequences of the nucleoprotein (N) genes of seven tospovirus isolates representing three serogroups were determined and used to establish phylogenetic parameters to delineate species within the Tospovirus genus of the Bunyaviridae. A high sequence divergence (55"
Twenty tomato spotted wilt virus (TSWV) isolates were serologically compared in ELISA employing five different procedures using a rabbit polyclonal antiserum against nucleocapsid proteins (NuAb R) and mouse monoclonal antibodies (MAbs), two directed to nucleocapsid proteins (N1 and N2) and four directed to glycoproteins G1 to G4. All the antisera were raised against TSWV-CNPH~. The 20 isolates were differentiated into two distinct serogroups. Serogroup I consisting of 16 isolates strongly reacted with NuAb R. The other four isolates were poorly recognized by NuAb ~ and were placed in another serogroup, designated II. The panel of MAbs differentiated the TSWV isolates into three serotypes. The 16 isolates forming serogroup I reacted strongly with the MAbs generated and were identified as serotype I isolates. The four isolates which made up serogroup II were split into serotypes II and III. The serotype II isolates did not respond or responded poorly with MAbs N1, N2 and G3. The two other isolates placed in serotype III were recognized by N 1 but not by N2 and G3. Two isolates became defective after several mechanical passages and failed to respond or responded very poorly with MAbs directed to glycoproteins. Our results show that ELISA employing polyclonal and monoclonal antisera is a useful tool to differentiate TSWV isolates and to detect defective forms. The results also strongly suggest that TSWV nucleocapsid proteins are less conserved than the glycoproteins.
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