César Augusto Diniz Xavier

Brazilian Begomovirus Populations Are Highly Recombinant, Rapidly Evolving, and Segregated Based on Geographical Location

ABSTRACT The incidence of begomovirus infections in crop plants sharply increased in Brazil during the 1990s following the introduction of the invasive B biotype of the whitefly vector, Bemisia tabaci. It is believed that this biotype transmitted begomoviruses from noncultivated plants to crop species with greater efficiency than indigenous B. tabaci biotypes. Either through rapid host adaptation or selection pressure in genetically diverse populations of noncultivated hosts, over the past 20 years various previously unknown begomovirus species have became progressively more prevalent in cultivated species such as tomato. Here we assess the genetic structure of begomovirus populations infecting tomatoes and noncultivated hosts in southeastern Brazil. Between 2005 and 2010, we sampled and sequenced 126 DNA-A and 58 DNA-B full-length begomovirus components. We detected nine begomovirus species in tomatoes and eight in the noncultivated host samples, with four species common to both tomatoes and noncultivated hosts. Like many begomoviruses, most species are obvious interspecies recombinants. Furthermore, species identified in tomato have probable parental viruses from noncultivated hosts. While the population structures of five well-sampled viral species all displayed geographical subdivision, a noncultivated host-infecting virus was more genetically variable than the four predominantly tomato-infecting viruses.

Synonymous site variation due to recombination explains higher genetic variability in begomovirus populations infecting non-cultivated hosts.

Begomoviruses are ssDNA plant viruses that cause serious epidemics in economically important crops worldwide. Non-cultivated plants also harbour many begomoviruses, and it is believed that these hosts may act as reservoirs and as mixing vessels where recombination may occur. Begomoviruses are notoriously recombination-prone, and also display nucleotide substitution rates equivalent to those of RNA viruses. In Brazil, several indigenous begomoviruses have been described infecting tomatoes following the introduction of a novel biotype of the whitefly vector in the mid-1990s. More recently, a number of viruses from non-cultivated hosts have also been described. Previous work has suggested that viruses infecting non-cultivated hosts have a higher degree of genetic variability compared with crop-infecting viruses. We intensively sampled cultivated and non-cultivated plants in similarly sized geographical areas known to harbour either the weed-infecting Macroptilium yellow spot virus (MaYSV) or the crop-infecting Tomato severe rugose virus (ToSRV), and compared the molecular evolution and population genetics of these two distantly related begomoviruses. The results reinforce the assertion that infection of non-cultivated plant species leads to higher levels of standing genetic variability, and indicate that recombination, not adaptive selection, explains the higher begomovirus variability in non-cultivated hosts.

Genetic structure of a Brazilian population of the begomovirus Tomato severe rugose virus (ToSRV)

Begomoviruses are whitefly-transmitted single-stranded DNA viruses of great economic importance in the tropics and subtropics. Several begomovirus species have been reported in tomatoes in Brazil, but only a few predominate in the field, for unknown reasons. In this study begomovirus-infected tomato samples were collected in Vicosa, State of Minas Gerais, in Nov/2009 and Dec/2010. Viral genomes were amplified, cloned and sequenced. A total of 36 DNA-A components were obtained. Sequence comparisons indicated the presence of a single begomovirus, Tomato severe rugose virus (ToSRV), with pairwise identities between isolates ranging from 97.3 to 100%. Subdivision tests indicated the existence of a single population. The analysis of variability descriptors indicated that the ToSRV population has a genetic variability similar to other begomovirus populations described in Brazil infecting tomato. Neutrality tests suggested the occurrence of purifying selection acting upon the population. Recombination analysis identified recombination events with begomoviruses from the weed species Sida micrantha. The wide distribution of ToSRV in the field and the detection of recombination indicate that continuous monitoring of viral populations in the field will be required to enable an efficient resistance-based control strategy for begomoviruses.

Begomovirus diversity in tomato crops and weeds in Ecuador and the detection of a recombinant isolate of rhynchosia golden mosaic Yucatan virus infecting tomato

Viral diseases caused by begomoviruses are of economic importance due to their adverse effects on the production of tropical and subtropical crops. In Ecuador, despite reports of significant infestations of Bemisia tabaci in the late 1990s, only very recently has a begomovirus, tomato leaf deformation virus (ToLDeV, also present in Peru), been reported in tomato. ToLDeV is the first monopartite begomovirus discovered that originated in the Americas, and its presence in Ecuador highlights the need for a wider survey of tomato-infecting begomoviruses in this country. Tomato and weed samples were collected in 2010 and 2011 in six provinces of Ecuador, and begomovirus genomes were cloned and sequenced using a rolling-circle-amplification-based approach. Most tomato samples from the provinces of Guayas, Loja, Manabi and Santa Elena were infected with tomato leaf deformation virus (ToLDeV). One sample from Manabi had a triple infection with ToLDeV, rhynchosia golden mosaic Yucatan virus (RhGMYuV) and an isolate that was a recombinant between the two. A new begomovirus was detected in another tomato sample from Manabi. Samples of Rhynchosia sp. from the provinces of Guayas and Manabi were infected by RhGMYuV. These results indicate not only the prevalence of ToLDeV in tomato in Ecuador but also the presence of other viruses, albeit at a much lower frequency.

The ever increasing diversity of begomoviruses infecting non-cultivated hosts: new species from Sida spp. and Leonurus sibiricus, plus two New World alphasatellites.

Begomoviruses (whitefly-transmitted, single-stranded DNA plant viruses) are among the most damaging pathogens causing epidemics in economically important crops worldwide. Besides cultivated plants, many weed and wild hosts act as virus reservoirs where recombination may occur, resulting in new species. The aim of this study was to further characterise the diversity of begomoviruses infecting two major weed genera, Sida and Leonurus. Total DNA was extracted from samples collected in the states of Rio Grande do Sul, Parana and Mato Grosso do Sul during the years 2009–2011. Viral genomes were enriched by rolling circle amplification (RCA), linearised into unit length genomes using various restriction enzymes, cloned and sequenced. A total of 78 clones were obtained: 37 clones from Sida spp. plants and 41 clones from Leonurus sibiricus plants. Sequence analysis indicated the presence of six bipartite begomovirus species and two alphasatellites. In Sida spp. plants we found Sida micrantha mosaic virus (SiMMV), Euphorbia yellow mosaic virus (EuYMV), and three isolates that represent new species, for which the following names are proposed: Sida chlorotic mottle virus (SiCMoV), Sida bright yellow mosaic virus (SiBYMV) and Sida golden yellow spot virus (SiGYSV), an Old World-like begomovirus. L. sibiricus plants had a lower diversity of begomoviruses compared to Sida spp., with only Tomato yellow spot virus (ToYSV) and EuYMV (for the first time detected infecting plants of the genus Leonurus) detected. Two satellite DNA molecules were found: Euphorbia yellow mosaic alphasatellite, for the first time detected infecting plants of the genus Sida, and a new alphasatellite associated with ToYSV in L. sibiricus. These results constitute further evidence of the high species diversity of begomoviruses in non-cultivated hosts, particularly Sida spp.

Genetic variability and population structure of the New World begomovirus Euphorbia yellow mosaic virus

The emergence of begomoviruses (whitefly-transmitted viruses classified in the genus Begomovirus, family Geminiviridae) in Brazil probably occurred by horizontal transfer from non-cultivated plants after the introduction of Bemisia tabaci MEAM1. The centre of diversity of Euphorbia heterophylla (Euphorbiaceae) is located in Brazil and Paraguay, where it is an invasive species in soybean and other crops. Reports of possible begomovirus infection of E. heterophylla in Brazil date back to the 1950s. In 2011, Euphorbia yellow mosaic virus (EuYMV) was described in symptomatic plants collected in the Brazilian state of Goiás. Here we assess the genetic variability and population structure of begomoviruses infecting E. heterophylla in samples collected throughout nine Brazilian states from 2009 to 2014. A total of 158 and 57 haplotypes were compared in DNA-A and DNA-B datasets, respectively. Analysis comparing population structure in a large sampled area enabled us to differentiate two subpopulations. Further, the application of discriminant analysis of principal components allowed the differentiation of six subpopulations according to sampling locations and in agreement with phylogenetic analysis. In general, negative selection was predominant in all six subpopulations. Interestingly, we were able to reconstruct the phylogeny based on the information from the 23 sites that contributed most to the geographical structure proposed, demonstrating that these polymorphisms hold supporting information to discriminate between subpopulations. These sites were mapped in the genome and compared at the level of amino acid changes, providing insights into how genetic drift and selection contribute to maintain the patterns of begomovirus population variability from a geographical structuring point of view.

Two new begomoviruses that infect non-cultivated malvaceae in Brazil

A high diversity of begomoviruses that infect non-cultivated plants has been noted in Brazil. Here, we report the complete sequences of two new species of bipartite begomoviruses from Sida sp. plants collected in the state of Piauí, northeastern Brazil. The genomes of these viruses show a genomic organization that is typical of New World begomoviruses. In phylogenetic analysis, two closely related viruses (sida angular mosaic virus, SiAMV and sida chlorotic vein virus, SiCVV) clustered with other begomoviruses described in tomato and Sida plants in Brazil. Evidence of recombination is shown among isolates of the species described.

Response of tomato ( Solanum L. section Lycopersicon Mill.) germplasm to begomovirus inoculation under controlled and field conditions

Ninety-five tomato accessions belonging to the Vegetable Crops Germplasm Bank of the Federal University of Viçosa (BGH-UFV) were evaluated in a protected environment (biolistic inoculation under greenhouse conditions) and in the field (natural infection) for resistance to bipartite begomoviruses present in Brazil. Plants were assessed by the visual evaluation of symptoms, and viral infection was confirmed by non-radioactive molecular hybridization. Univariate statistical analyses and correlations were made between the traits. High and significant correlations were found between the percentages of visual symptoms and of viral replication, with values of 0.67 in the protected environment and 0.60 in the field. We concluded that under the protected environment, the accessions BGH-2144, BGH-2150, BGH-6878 and BGH-6881 displayed resistance to infection. Under field conditions with natural infection, the best results were obtained for the accessions BGH-2080 and BGH-6881. Only the BGH-6881 (Solanum peruvianum L.) accession excelled in both conditions; this resistance was attributed to the presence of the Ty-2 and Ty-3 resistance gene alleles in heterozygosity. In general, all of the accessions selected in both experimental conditions can serve as sources for the development of cultivars tolerant or resistant to the bipartite begomoviruses present in Brazil.

Complete genome sequences of two gemycircularviruses associated with non-cultivated plants in Brazil

Gemycircularviruses (genus Gemycircularvirus, family Genomoviridae) are single-stranded DNA viruses that are spread around the world in association with several organisms and environments. In this work, we identified two gemycircularviruses associated with two non-cultivated plants in Brazil, Momordica charantia and Euphorbia heterophylla. Both viruses display the general genome structure of gemycircularviruses. The virus isolated from M. charantia showed the highest nucleotide sequence identity with Pteropus associated gemycircularvirus 5, and an atypical structure consisting of a hairpin embedded in the major stem-loop was observed in the intergenic region. The virus from E. heterophylla showed the highest nucleotide sequence identity with Odonata associated gemycircularvirus 1. Phylogenetic analysis groups the two new viruses together with other genomoviruses of the genus Gemycircularvirus.

CARACTERIZAÇÃO DA RESISTÊNCIA DE SUBAMOSTRAS DE TOMATE DO BGH-UFV AO GEMINIVÍRUS TOMATO YELLOW SPOT VIRUS

The viruses transmitted by whiteflies are among those causing relevant losses in tomato cultivation. Among the measures to control these agents, introducing genes for resistance constitutes the main control measure, together with vector control. The objective of this work was to screen for sources of natural resistance to Tomato yellow spot virus (ToYSV) in S. lycopersicum germplasm from the Banco de Germoplasma de Hortalicas (BGH) of the Universidade Federal de Vicosa (UFV), Minas Gerais, Brazil. The 99 accessions and two susceptible controls were inoculated using biolistics. Inoculated plants were grown under greenhouse conditions. The percentage of plants displaying virus symptoms was evaluated at 10, 20 and 30 days after inoculation (DAI). Virus presence or absence in the inoculated plants was confirmed by hybridization with probes labeled with a-[32P]-dCTP, for each evaluation date. Inoculated plants produced typical disease symptoms showing different behavior on the genotypes in relation to ToYSV. Some of the evaluated genotypes showed higher virus tolerance compared to two susceptible controls, in particular the accessions BGH-2039V and BGH-2041 which showed no symptoms and no viral DNA accumulation in 80% of the inoculated plants at 30 DAI. The results suggest that the selected tomato accesses are good sources of resistance to new tomato cultivars tolerant to ToYSV.