eThe 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.

The incidence of tomato-infecting begomoviruses has sharply increased in Brazil following the introduction of the B biotype of the whitefly vector in the early 1990s. Five definitive species and six tentative species have been described since then. Here, we report the detection of members of an additional six novel species, three in tomato and three infecting weeds that are commonly associated with tomato fields: Blainvillea rhomboidea, Sida rhombifolia and Sida micrantha. Tomato and weed samples were collected in two major tomato-growing regions of southeastern Brazil in 2005 and 2007. Two of the novel viruses were present in tomato plants collected in Paty do Alferes, Rio de Janeiro state. Three novel viruses were present in weed samples collected in Coimbra, Minas Gerais state. One virus was present in tomato samples collected at both locations. Genome features indicate that all six species are typical New World, bipartite begomoviruses. However, the viruses belonging to two of the novel species did not cluster with the Brazilian viruses in a phylogenetic tree. These species could represent a distinct lineage of New World begomoviruses, found in Brazil for the first time.

Begomoviruses (single-stranded DNA plant viruses) are responsible for serious agricultural threats. Begomovirus populations exhibit a high degree of within-host genetic variation and evolve as quickly as RNA viruses. Although the recombination-prone nature of begomoviruses has been extensively demonstrated, the relative contribution of recombination and mutation to the genetic variation of begomovirus populations has not been assessed. We estimated the genetic variability of begomovirus datasets from around the world. An uneven distribution of genetic variation across the length of the cp and rep genes due to recombination was evident from our analyses. To estimate the relative contributions of recombination and mutation to the genetic variability of begomoviruses, we mapped all substitutions over maximum likelihood trees and counted the number of substitutions on branches which were associated with recombination (ηr) and mutation (ημ). In addition, we also estimated the per generation relative rates of both evolutionary mechanisms (r/μ) to express how frequently begomovirus genomes are affected by recombination relative to mutation. We observed that the composition of genetic variation in all begomovirus datasets was dominated by mutation. Additionally, the low correlation between the estimates indicated that the relative contributions of recombination and mutation are not necessarily a function of their relative rates. Our results show that, although a considerable fraction of the genetic variation levels could be assigned to recombination, it was always lower than that due to mutation, indicating that the diversification of begomovirus populations is predominantly driven by mutational dynamics.

A survey of begomoviruses infecting leguminous weeds (family Fabaceae) was carried out in four states of northeastern Brazil. A total of 26 full-length begomovirus components (19 DNA-A and seven DNA-B, with three pairs of cognate A and B components) were amplified using rolling-circle amplification, then cloned and sequenced. Sequence analysis indicated the presence of six species, four of them novel. In phylogenetic analysis five of the viruses clustered with other Brazilian begomoviruses, but one of them (Euphorbia yellow mosaic virus, EuYMV) clustered with viruses from other countries in Central and South America. Evidence of recombination was found among isolates of Macroptilium yellow spot virus (MaYSV). The MaYSV population had a high degree of genetic variability. Macroptilium lathyroides was revealed as a common host for several of these viruses, and could act as a mixing vessel from which recombinant viruses could emerge. The results indicate that leguminous weeds are reservoirs of several begomoviruses in Brazil, and could play a significant role in begomovirus epidemics, both as inoculum sources and as sources of emerging novel viruses.

The biological and molecular characterization of six isolates of a new Cowpea mild mottle virus strain (CPMMV; Carlavirus, Betaflexiviridae) are reported. Soybean plants with mosaic and stem necrosis were collected in Bahia, Goi as, Mato Grosso and Minas Gerais states, Brazil. Complete genomes of the CPMMV isolates are 8180-8198 nucleotides (nt) long, excluding the 3′-polyadenylated tail, and have 67-68% nt sequence identity with a Ghana isolate of CPMMV, the only CPMMV isolate for which the genome has previously been sequenced. The replicase has only 60-61% nt sequence identity with the Ghana CPMMV isolate, and the coat protein (CP) is highly conserved (79% nt sequence identity and 95-96% amino acid sequence identity). The high CP identity and the phylogenetic analyses supported the classification of the Brazilian isolates as CPMMV. Biological and molecular differences with the Ghana CPMMV isolate were found and indicated that the six isolates represent a distinct CPMMV strain denominated as CPMMV-BR. Furthermore, it is shown that recombination occurred mainly in the polymerase gene, and may occur less frequently in other regions of the CPMMV genome.

Diseases caused by begomoviruses are a serious constraint to crop production in many tropical and subtropical areas of the world, including Brazil. Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that are often associated with weed plants, which may act as natural reservoirs of viruses that cause epidemics in crop plants. Cleome affinis (family Capparaceae) is an annual weed that is frequently associated with leguminous crops in Brazil. Samples of C. affinis were collected in four states in the northeast of Brazil. Analysis of 14 full-length DNA-A components revealed that only one begomovirus was present, with 91-96% identity to cleome leaf crumple virus (ClLCrV). In a phylogenetic tree, ClLCrV forms a basal group relative to all other Brazilian begomoviruses. Evidence of multiple recombination events was detected among the ClLCrV isolates, which also display a high degree of genetic variability. Despite ClLCrV being the only begomovirus found, its phylogenetic placement, high genetic variability and recombinant nature suggest that C. affinis may act as a source of novel viruses for crop plants. Alternatively, ClLCrV could be a genetically isolated begomovirus. Further studies on the biological properties of ClLCrV should help to clarify the role of C. affinis in the epidemiological scenario of Brazilian begomoviruses.

BackgroundBegomoviruses are dicot-infecting, whitefly-transmitted viruses with a genome comprised of one or two molecules of circular, single-stranded DNA. In Brazil, tomato-infecting begomoviruses have emerged as serious pathogens since the introduction of a new biotype of the insect vector in the mid-1990’s. Tomato rugose mosaic virus (ToRMV) and Tomato severe rugose virus (ToSRV) are often found in tomato fields. The complete sequence of the DNA-B components of ToSRV and ToRMV show an identity of 98.2%. Additionally, the high nucleotide identity (96.2%) between their common regions indicates that these two viruses may share the same DNA-B.MethodsTomato seedlings were biolistically inoculated with ToSRV (DNA-A and DNA-B) and ToRMV (DNA-A and DNA-B) infectious clones in every possible combination of single or mixed infection. Symptom expression was evaluated for up to 35 days post-inoculation (dpi). DNA was extracted at 28 dpi and the presence of each viral genomic component was examined by rolling circle amplification (RCA) followed by digestion, as well as by quantitative, real-time PCR. Sequence comparisons, recombination and phylogenetic analyzes were performed using EMBOSS needle, RDP program and maximum likelihood inference, respectively.ResultsSymptoms in tomato plants inoculated with the different combinations of ToRMV and ToSRV DNA-A and DNA-B components consisted of a typical mosaic in all combinations. Pseudorecombinants were formed in all possible combinations. When two DNA-A or two DNA-B components were inoculated simultaneously, the ToRMV components were detected preferentially in relation to the ToSRV components. The combination of minor changes in both the Rep protein and the CR may be involved in the preferential replication of ToRMV components. Recombination and phylogenetic analyzes support the exchange of genetic material between ToRMV and ToSRV.ConclusionsToRMV and ToSRV form viable pseudorecombinants in their natural host (Solanum lycopersicum) and share the same DNA-B. ToRMV DNA components are preferentially replicated over ToSRV components. These results indicate that the emergence of ToRMV involved both recombination and pseudorecombination, further highlighting the importance of these mechanisms in the emergence and adaptation of begomoviruses.

RESUMOViroses causadas por vírus do gênero Badnavirus são responsáveis por grandes prejuízos à cultura do inhame no Nordeste brasileiro. O conhecimento da variabilidade destes patógenos pode fornecer informações importantes sobre seu potencial evolutivo, permitindo a elaboração de melhores estratégias de manejo da doença. A análise de 425 amostras foliares de inhame coletadas em três estados do Nordeste brasileiro, em 2010, revelou uma alta incidência (93,3%) de badnaviroses. Para avaliar a variabilidade genética dos badnavírus infectando inhame, um fragmento de 579 nucleotídeos correspondente à região codificante da transcriptase reversa (RT)/RNaseH dos isolados amostrados foi amplificada por PCR e sequenciada. A análise filogenética das sequências de nucleotídeos revelou que os isolados dividem-se em dois grupos. Um é altamente relacionado com Dioscorea bacilliform AL virus (DBALV), enquanto o outro forma um clado altamente divergente dentro do gênero Badnavirus. Os isolados de DBALV apresentam 70-98% de identidade nucleotídica entre si e foram detectados em todas as áreas avaliadas e em D. alata e D. cayennensis, as duas espécies de inhame mais cultivadas no Nordeste. Os isolados do outro grupo compartilham 47-58% de identidade com isolados de DBALV e 78-95% entre si e foram encontrados apenas em D. alata na Paraíba. Palavras-chave: Dioscorea bacilliform AL virus, RT/RNaseH, sequências endógenas. ABSTRACT Genetic variability of badnavirus isolates infecting yam (Dioscorea spp.) in northeastern BrazilDiseases caused by viruses of the genus Badnavirus are responsible for great losses in yam crops in northeastern Brazil. Knowledge of pathogen variability can provide important information about its evolutionary potential and may allow for development of better strategies for disease management. The analysis of 425 leaf samples of yam obtained in 2010 in three states of northeastern Brazil revealed a high incidence (93.3%) of badnaviruses. To evaluate the variability of yam-infecting badnaviruses, a 579-nucleotide fragment corresponding to the reverse transcriptase (RT)/RNaseH coding region was PCR-amplified and directly sequenced. Phylogenetic analysis of nucleotide sequences revealed that the isolates can be divided into two groups. The first group is highly related to Dioscorea bacilliform AL virus (DBALV), while the other set of sequences formed a highly divergent clade within the genus Badnavirus. The DBALV isolates have 70-98% nucleotide sequence identity with each other. DBALV was detected in all areas assessed and in the two most cultivated species of yam in the northeast (D. alata and D. cayanensis). The other group shares 47-58% nucleotide sequence identity with the DBALV isolates and 78-95% amongst themselves, and was found only in D. alata in the state of Paraíba. Key words: Dioscorea bacilliform AL virus, endogenous sequences, RT/RNaseH. O inhame (Dioscorea spp.) é uma cultura de importância econômica e social nas regiões tropicais, incluindo o nordeste do Brasil que produziu 38.256 toneladas em 2006(...