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Keywords:

  • Fabaceae;
  • geminivirus;
  • Macroptilium;
  • MaYSV;
  • recombination

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Weeds and other wild hosts, whether indigenous or introduced, can be infected by a large number of plant viruses, and therefore may play a role as reservoirs of crop-infecting viruses (Seal et al., 2006). Furthermore, viral populations in weeds may be subject to distinct selective constraints compared to those in crop species, since the genetic base of wild species is generally wider than that of crop species (Webster et al., 2007). Knowledge of the dynamics of genetic variability in reservoir hosts is therefore essential to understand how viral populations evolve, with obvious implications for the durability of disease management strategies based on genetic resistance (Seal et al., 2006).

Viruses belonging to the family Geminiviridae have a genome comprised of circular ssDNA molecules encapsidated in twinned quasi-icosahedral particles. Based on their genome organization, host range and insect vectors, geminiviruses are classified into four different genera: Mastrevirus, Topocuvirus, Curtovirus and Begomovirus (Fauquet et al., 2008). Begomoviruses are whitefly-transmitted geminiviruses that constitute one of the most economically important groups of plant viruses because of their high incidence and the severity of diseases they cause in vegetable and field crops throughout tropical and subtropical regions of the world (Morales & Anderson, 2001). In South America, begomoviruses are limiting factors to tomato (Solanum lycopersicum), common bean (Phaseolus vulgaris) and, to a lesser extent, sweet and hot pepper (Capsicum spp.) production (Morales, 2006). The most severely affected crops in Brazil are beans and tomato (Faria et al., 2000). In beans (P. vulgaris and P. lunatus), golden mosaic caused by Bean golden mosaic virus (BGMV) has been an important disease since the 1970s, and its dissemination has been attributed to the increase in soyabean cultivation (Costa, 1976). In tomato, the emergence of begomovirus-associated diseases coincided with the introduction and spread of the B biotype of Bemisia tabaci during the mid-1990s (Ribeiro et al., 1998).

Begomoviruses are also associated with a wide range of weed species, which in some cases act as primary inoculum sources for crop plants (Frischmuth et al., 1997). Most of the weed species commonly reported as begomovirus hosts belong to the families Euphorbiaceae, Fabaceae, Malvaceae and Solanaceae (Morales & Anderson, 2001). Surveys carried out to identify weed-associated begomoviruses in Brazil indicate that, similar to what is observed for begomoviruses in crops, a very high species diversity is present (Ambrozevicius et al., 2002; Castillo-Urquiza et al., 2008; Paprotka et al., 2010b). However, information on the genetic variability of begomoviruses in weed and other wild hosts is lacking.

It is believed that begomoviruses infecting wild hosts in Brazil have been horizontally transferred to crop plants, and that in the new host they can adapt by mutation, recombination or pseudorecombination, eventually emerging as novel species (Castillo-Urquiza et al., 2008; Fernandes et al., 2009). Four independent lines of evidence give support to this hypothesis. First, all begomoviruses reported so far in crops in Brazil are indigenous to the country, and except for neighbouring Argentina (Rodríguez-Pardina et al., 2010) have not been reported elsewhere. Secondly, the biological characterization of a number of crop-infecting begomoviruses (e.g. BGMV; Tomato chlorotic mottle virus, ToCMoV; Tomato rugose mosaic virus, ToRMV; Tomato yellow spot virus, ToYSV; and Tomato yellow vein streak virus, ToYVSV) indicated that they can also infect weeds such as Datura stramonium, Macroptilium lathyroides, Nicandra physaloides and Solanum nigrum (Chagas et al., 1981; Fernandes et al., 2006; Calegario et al., 2007; Ribeiro et al., 2007). Thirdly, begomoviruses originally detected in wild plants, such as Sida mottle virus (SiMoV) and Sida micrantha mosaic virus (SiMMV), have been found naturally infecting crop species (Castillo-Urquiza et al., 2007, 2010). Fourthly, evidence of recombination and pseudorecombination has been obtained for the viruses which are prevalent in crop species such as tomato and common bean (Andrade et al., 2006; Inoue-Nagata et al., 2006; Ribeiro et al., 2007). The purpose of this study was to characterize begomovirus populations infecting leguminous weeds (family Fabaceae) in northeastern Brazil, as a step towards assessing their role as begomovirus reservoirs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Sample collection, processing and storage

Surveys of leguminous weeds were carried out in locations throughout the states of Alagoas (AL), Paraíba (PB), Pernambuco (PE) and Sergipe (PE) (Fig. S1). Plants displaying symptoms of mosaic, yellowing and stunting typical of begomovirus infection were preferentially collected. Samples were desiccated by pressing and stored at room temperature.

DNA amplification and cloning

DNA extraction was carried out from dried leaves according to Doyle & Doyle (1987). To confirm the presence of begomoviruses, PCR was carried out using universal begomovirus primers (Rojas et al., 1993). Full-length viral genomes were amplified from PCR-positive samples by rolling-circle amplification (RCA) (Inoue-Nagata et al., 2004), cloned in pBLUESCRIPT KS+ (Stratagene) after monomerization with the restriction enzymes BamHI, ClaI, EcoRI, HindIII, KpnI, PstI, SacI or SpeI, and sequenced commercially (Macrogen Inc.) by primer walking.

Sequence comparisons and phylogenetic analysis

DNA-A nucleotide sequences were initially submitted to a blast search for preliminary species assignment based on the 89% threshold level established by the Geminiviridae Study Group of the ICTV (Fauquet et al., 2008). Additional pairwise nucleotide sequence comparisons were made with DNAMan v. 4.0 using the Optimal Alignment option with the following parameters: Ktuple = 2, Gap penalty = 7, Gap open = 10, Gap extension = 5. Nucleotide sequences of begomoviruses used in the recombination and phylogenetic analyses were aligned using the muscle module in mega v. 5.0 (Tamura et al., 2011). Phylogenetic analysis was performed using Bayesian inference and Markov chain Monte Carlo (MCMC) simulation implemented in MrBayes v. 3.0 (Ronquist & Huelsenbeck, 2003). Bayesian analysis was conducted on the aligned data set after the nucleotide substitution model was determined by MrModeltest v. 2.2 (Nylander, 2004). The MCMC analysis of four chains started with a heating parameter of 0·1 from a random tree topology and lasted 5 000 000 generations. Trees were saved each 100 generations, resulting in 50 000 saved trees. Burn-in was set at 1 250 000 generations, after which the likelihood values were stationary, leaving 37 000 trees from which the 50% majority rule consensus trees and posterior probabilities were calculated.

Recombination analysis

Phylogenetic network analysis for evidence of recombination was performed with the neighbour-net method implemented in the program SplitsTree4 (Huson & Bryant, 2006). Analysis of potential recombination events was carried out using the recombination detection program rdp v. 3.0 (Martin et al., 2010) using default parameters.

Genetic variability of the MaYSV population

The partition of genetic variability and inferences about population structure were based on Wright’s F fixation index (Weir, 1996). The ΦST parameter was calculated with the program arlequin 3.11 (Excoffier et al., 2005), using the Kimura 2-parameter distance and estimating statistical significance by permutation analysis with 1000 replications.

The main descriptors of genetic variability were quantified: number of polymorphic sites, total number of mutations (η), average number of nucleotide differences (k), nucleotide diversity (π), number of haplotypes, haplotype diversity (Hd), and Watterson’s estimate of the population mutation rate based on the total number of segregating sites (θ-w) and on the total number of mutations (θ-Eta). Four types of neutrality tests were used to test the hypothesis of occurrence of selection in populations: Tajima’s D, Fu and Li’s D* and F* and the test based on the number of synonymous (Ds) and non-synonymous (Dns) substitutions with the Pamilo-Bianchi-Li (PBL) model. These analyses were performed using the program DnaSP v. 5 (Rozas et al., 2003).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Sequence comparisons and phylogenetic analysis

Weed samples belonging to the genera Canavalia, Calopogonium, Centrosema and Macroptilium (all in the family Fabaceae), displaying typical symptoms of begomovirus infection (Fig. 1), were collected in four states of northeastern Brazil in May 2009 and July 2010 (Table 1). All 24 samples tested positive for the presence of begomoviruses by PCR (data not shown). From these samples, 19 full-length DNA-A components and seven DNA-B components were cloned, including three pairs of cognate A and B components (Table 1). blast analysis and pairwise sequence comparisons of the DNA-A clones indicated the presence of six begomovirus species (Table 1).

image

Figure 1.  Symptoms in leguminous weeds infected by three novel begomoviruses in northeastern Brazil. (a) Yellow spot symptoms in the sample of Macroptilium lathyroides from which isolate BR:Mac1:10 (Macroptilium yellow spot virus, MaYSV) was obtained. (b) Yellow mosaic and vein banding symptoms in the sample of Macroptilium sp. from which isolate BR:Mac4:10 (Macroptilium yellow vein virus, MaYVV) was obtained. (c) Reticulate yellow mosaic in the sample of M. lathyroides from which isolate BR:Mur1:09 (Macroptilium yellow net virus, MaYNV) was obtained.

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Table 1.   Full-length clones corresponding to bipartite begomovirus DNA-A and DNA-B components obtained from samples of leguminous weeds collected in the Brazilian northeastern states of Alagoas (AL), Paraíba (PB), Pernambuco (PE) and Sergipe (SE)
HostLocationYearCloneSpecies assignmentaGenBank acc. no.
DNA-ADNA-B
  1. aSpecies assignment based on the ICTV-established criteria of 89% nucleotide sequence identity for the full-length DNA-A (Fauquet et al., 2008). MaYNV, Macroptilium yellow net virus; EuYMV, Euphorbia yellow mosaic virus; CenYSV, Centrosema yellow spot virus; BGMV, Bean golden mosaic virus; MaYSV, Macroptilium yellow spot virus; MaYVV, Macroptilium yellow vein virus; TCrLYV, Tomato crinkle leaf yellows virus. Underlined species are reported for the first time in this study.

  2. bTentative assignment, since the cognate DNA-A was not cloned.

Macroptilium lathyroidesMurici, AL2009BR:Mur1:09ABR:Mur1:09BMaYNVJN418998, JN418999
M. atropurpureumCaruaru, PE2009BR:Car2:09ABR:Car2:09BEuYMVJN419000, JN419001
Centrosema brasilianumCaruaru, PE2009BR:Car1:09A CenYSVJN419002
M. lathyroidesCaruaru, PE2010BR:Car3:10A BGMVJN419003
M. lathyroidesCaruaru, PE2010BR:Car4:10A BGMVJN419004
M. lathyroidesBarra de Santana, PB2009 BR:Bas1:09BMaYSVJN419005
M. lathyroidesMaceió, AL2010BR:Mac2:10A BGMVJN419006
M. lathyroidesCedro, SE2009BR:Ced1:09A MaYSVJN419007
M. lathyroidesMessias, AL2010 BR:Mes2:10BBGMVbJN419008
M. lathyroidesMaceió, AL2010BR:Mac1:10A MaYSVJN419009
M. atropurpureumMaceió, AL2010 BR:Mac3:10BTCrLYVbJN419010
M. atropurpureumQuipapá, PE2010 BR:Qui1:10BTCrLYVbJN419011
M. lathyroidesBatalha, AL2010BR:Bat1:10A MaYSVJN419012
M. lathyroidesÁgua das Flores, AL2010BR:Agf1:10A MaYSVJN419013
M. lathyroidesÁgua das Flores, AL2010BR:Agf2:10A MaYSVJN419014
Calopogonium mucunoidesPiranhas, AL2010BR:Pir1:10A MaYSVJN419015
Calopogonium mucunoidesDelmiro Gouveia, AL2010BR:Deg1:10A MaYSVJN419016
M. lathyroidesDelmiro Gouveia, AL2010 BR:Deg2:10BBGMVbJN419017
M. lathyroidesInhapi, AL2010BR:Inp1:10A MaYSVJN419018
Canavalia sp.Inhapi, AL2010BR:Inp2:10A MaYSVJN419019
M. lathyroidesPalmeira dos Índios, AL2010BR:Pdi1:10A MaYSVJN419020
Macroptilium sp.Maceió, AL2010BR:Mac4:10A MaYVVJN419021
M. lathyroidesMaceió, AL2010BR:Mac5:10A MaYSVJN419022

Clones BR:Car2:09A and BR:Car2:09B from Macroptilium atropurpureus corresponded to an isolate of Euphorbia yellow mosaic virus (EuYMV), with 97% identity with the DNA-A of a EuYMV isolate from Brazil (FJ619507). Clones BR:Car3:10A, BR:Car4:10A and BR:Mac2:10A obtained from M. lathyroides corresponded to isolates of BGMV, with 89–90% identity with the type isolate from common bean (M88686). One isolate, represented by clones BR:Mur1:09A and BR:Mur1:09B from M. lathyroides, was representative of a new species which was most closely related to ToCMoV (AF490004, 86% identity), and for which the name Macroptilium yellow net virus (MaYNV) is proposed. Clone BR:Car1:09A from Centrosema brasilianum also represented a new species, for which the name Centrosema yellow spot virus (CenYSV) is proposed. A third new species, for which the name Macroptilium yellow spot virus (MaYSV) is proposed, was represented by clones BR:Agf1:10A, BR:Agf2:10A, BR:Bas1:09A and BR:Bas1:09B, BR:Bat1:10A, BR:Ced1:09A, BR:Inp1:10A, BR:Mac1:10A, BR:Mac5:10A and BR:Pdi1:10A (from M. lathyroides), BR:Deg1:10A and BR:Pir1:10A (from Calopogonium mucunoides), and BR:Inp2:10A (from Canavalia sp.). A fourth novel species was represented by clone BR:Mac4:10A from M. lathyroides, and the name Macroptilium yellow vein virus (MaYVV) is proposed for this species.

The genomes of all four new species had a typical bipartite, New World begomovirus organization, with five ORFs in the DNA-A and two in the DNA-B (Table S1). Isolates of the four new species displayed <85% DNA-A sequence identity amongst them (Table S2). The common regions (CR) had the conserved nonanucleotide (5′-TAATATT//AC-3′) as part of a stem-loop in the origin of replication. Cognate DNA-A and DNA-B components (BR:Mur1:09A/B, MaYNV; and BR:Bas1:09A/B, MaYSV) had identical iterons, but they differed among the four species: GGAGT for CenYSV, GGAG for MaYSV and GGTG for MaYNV and MaYVV.

A phylogenetic tree based on the complete DNA-A nucleotide sequence of the begomoviruses from leguminous weeds and other Brazilian begomoviruses was constructed used Bayesian inference, with the nucleotide substitution model GTR+I+G (Fig. 2). The weed-infecting begomoviruses were placed in three major monophyletic clusters within the tree. The first cluster, with 98% Bayesian posterior probability (Bpp), included the EuYMV and BGMV isolates (BR:Car2:09, BR:Car3:10, BR:Car4:10 and BR:Mac2:10), the novel species CenYSV (BR:Car1:09) and MaYVV (BR:Mac4:10), and other bean-, tomato- and weed-infecting begomoviruses. Within this major cluster, CenYSV grouped with tomato- and weed-infecting begomoviruses, and MaYVV grouped with BGMV. The second major cluster, with 100% Bpp, included the new species MaYSV (BR:Bas1:09 plus 11 additional isolates) and Blainvillea yellow spot virus (BlYSV), also a weed-infecting begomovirus. The third major cluster, with 100% Bpp, comprised the novel species MaYNV (BR:Mur1:09) and ToCMoV.

image

Figure 2.  Bayesian 50% majority rule consensus tree of begomoviruses from leguminous weeds and other Brazilian begomoviruses. The four novel species detected in leguminous weeds (CenYSV, Centrosema yellow spot virus; MaYVV, Macroptilium yellow vein virus; MaYSV, Macroptilium yellow spot virus; and MaYNV, Macroptilium yellow net virus) are indicated in bold face. The 12 isolates belonging to MaYSV are underlined. Numbers at the nodes indicate Bayesian posterior probabilities. AbBV, Abutilon Brazil virus; BGMV, Bean golden mosaic virus; BlYSV, Blainvillea yellow spot virus; ClLCrV, Cleome leaf crumple virus; EuYMV, Euphorbia yellow mosaic virus; NDNV, Nicandra deforming necrosis virus; OMoV, Okra mottle virus; PSLDV, Passionfruit severe leaf distortion virus; SiCmMV, Sida common mosaic virus; SiMBV, Sida mosaic Brazil virus; SiMMV, Sida micrantha mosaic virus; SiMoV, Sida mottle virus; SiYLCV, Sida yellow leaf curl virus; SiYMV, Sida yellow mosaic virus; SoBlMV, Soybean blistering mosaic virus; ToCMoV, Tomato chlorotic mottle virus; ToCmMV, Tomato common mosaic virus; TGMV, Tomato golden mosaic virus; ToMlMV, Tomato mild mosaic virus; ToRMV, Tomato rugose mosaic virus; ToSRV, Tomato severe rugose virus; ToYSV, Tomato yellow spot virus; ToYVSV, Tomato yellow vein streak virus.

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A second phylogenetic tree based on the complete DNA-A sequences of viruses from leguminous weeds and other begomoviruses from the Americas was constructed (Fig. S2). The viruses within this tree clustered into four major groups. Clusters 1 and 4 comprised only non-Brazilian viruses. Cluster 2 included EuYMV, two weed-infecting viruses from Brazil (Sida yellow leaf curl virus, SiYLCV; and Tomato common mosaic virus, ToCmMV), plus several viruses from other countries in the Americas. The other five begomoviruses from leguminous weeds were grouped in cluster 3, which mainly comprised Brazilian begomoviruses that infect bean, tomato, passionfruit and weeds.

Recombination analysis

Phylogenetic relationships inferred by neighbour-net analysis based on a data set consisting of all Brazilian begomoviruses, including the viruses from leguminous weeds described in this work, revealed clear evidence of multiple recombination events (Fig. 3). Strong evidence for recombination was found in the cluster containing the 12 MaYSV clones. These results were confirmed using a second data set comprised only of the viruses from leguminous weeds (Fig. S3). Evidence for recombination was again obtained when the analysis was restricted to the 12 MaYSV clones (Fig. S4), and was reinforced by phylogenetic inconsistency observed for BR:Bat1:10A, BR:Pir1:10A and BR:Inp1:10A, which always grouped separately from the other nine isolates (Figs S3 and S4).

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Figure 3.  Phylogenetic evidence for recombination among Brazilian begomoviruses, including those described in this work. Neighbour-net network analysis was performed using SplitsTree4. Formation of a reticular network rather than a bifurcated tree indicates the occurrence of recombination. Virus names and accession numbers are as in Fig. 2.

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To further investigate these putative recombination signals, the same three sets of sequences were analysed using the rdp3 package. This analysis identified many unique recombination signals. To exclude unreliable signals, only recombination events supported by at least four different methods were selected. A strongly supported recombination event was detected involving MaYSV clones BR:Bas1:09A and BR:Mac1:10A, with breakpoints at the CP gene. This event was detected with all three data sets (event four; Tables 2, S3 and S4), with MaYNV (BR:Mur1:09A) identified as one of the putative parents (Table 2). A recombination event also with breakpoints at the CP gene was identified for MaYSV clones BR:Bat1:10A, BR:Inp1:10A and BR:Pir1:10A with the three data sets (event 6; Tables 2, S3 and S4), with possible parents varying depending on the data set: BR:Agf2:10A when only MaYSV isolates were analysed (Table S3), CenYSV (BR:Car1:09A) when all viruses from leguminous weeds were included (Table S4), and BGMV (BR:Mac2:10A) when all Brazilian begomoviruses were included (Table 2). A recombination event in the Rep gene was detected for the three BGMV clones (BR:Car3:10A, BR:Car4:10A and BR:Mac2:10A) and MaYVV (BR:Mac4:10A), with one of the parents identified as SiBV or CenYSV (event 3; Tables 2 and S4). Another recombination event was observed in the Rep gene of MaYSV clones BR:Agf1:10A, BR:Agf2:10A, BR:Bas1:09A, BR:Ced1:09A, BR:Deg1:10A, BR:Inp2: 10A, BR:Mac1:10A and BR:Pdi1:10A, with BlYSV identified as one of the parents (event 5; Table 2).

Table 2.   Putative recombination events detected among begomoviruses infecting leguminous weeds in northeastern Brazil
EventSpecies-isolateParentsaBreakpointsbP-value
InitialFinalRcGBMCS3S
  1. aWhen only the major parent is indicated, the minor parent has not been identified. ‘Unknown’, neither parent identified.

  2. bNumbering starts at the first nucleotide after the cleavage site at the origin of replication and increases clockwise.

  3. cR, rdp; G, GeneConv; B, Bootscan; M, MaxChi; C, chimaera; S, SisScan; 3S, 3seq.

  4. d−, no recombination event detected.

  5. eLocation of breakpoints varies slightly for each isolate.

1EuYMV-[Car2:09]Unknown184121241 × 10−22d2 × 10−173 × 10−113 × 10−93 × 10−3
2CenYSV-[Car1:09]Unknown215023853 × 10−76 × 10−73 × 10−55 × 10−51 × 10−42 × 10−4
3BGMV-[Car3:10], [Car4:10], [Mac2:10], MaYVV-[Mac4:10]SiBV1950e25371 × 10−71 × 10−28 × 10−52 × 10−72 × 10−52 × 10−9
4MaYSV-[Bas1:09], [Mac1:10]MaYNV457e9026 × 10−112 × 10−43 × 10−116 × 10−74 × 10−78 × 10−56 × 10−11
5MaYSV-[Agf1:10], [Agf2:10], [Bas1:09], [Ced1:09], [Deg1:10], [Inp2:10], [Mac1:10], [Pdi1:10]BlYSV1787e25762 × 10−81 × 10−28 × 10−73 × 10−71 × 10−52 × 10−136 × 10−4
6MaYSV-[Bat1:10], [Inp1:10], [Pir1:10]BGMV481e6092 × 10−21 × 10−28 × 10−12 × 10−22

Genetic variability of the MaYSV population

Population analysis indicated that the 12 MaYSV isolates comprised a single population, with no subdivision based on geographical location, year of collection or host.

The MaYSV population had a high degree of genetic variability, as indicated by genetic descriptors with considerably higher values than those observed for two populations of tomato-infecting begomoviruses from southeastern Brazil (Table 3; Castillo-Urquiza et al., 2010).

Table 3.   Genetic structure of a population of Macroptilium yellow spot virus (MaYSV) obtained from leguminous weeds in northeastern Brazil
PopulationNumber of sequencesDNA-A length (nt)sbEtackdπehfHdgθ-whθ-Etai
  1. bTotal number of segregating sites.

  2. cTotal number of mutations.

  3. dAverage number of nucleotide differences between sequences (Tajima’s estimate of the population mutation rate, θ).

  4. eNucleotide diversity.

  5. fHaplotype number.

  6. gHaplotype diversity.

  7. hWatterson’s estimate of the population mutation rate based on the total number of segregating sites.

  8. iWatterson’s estimate of the population mutation rate based on the total number of mutations.

MaYSV122658402419150·1770·0572121·00·05370·0542
ToCmMVa22256010310436·6450·0143200·9870·01100·0111
ToYVSVa26256249495·3810·0021250·9970·00500·0050

Neutrality tests were used to assess for evidence of selection or demographic forces acting on the MaYSV population. The four ORFs encoded by the DNA-A (Rep, Trap, Ren and CP) varied in this regard. Negative values were obtained, but were not statistically supported, for Tajima’s D, Fu and Li’s D and Fu and Li’s F for Ren, Trap and CP (Table 4). The Rep ORF showed positive values for these three tests, confirming the hypothesis of neutrality. The values of dN/dS <1 for all ORFs are indicative of purifying selection acting on this population.

Table 4.   Results of four neutrality tests for each open reading frame (ORF) in the DNA-A of viral isolates comprising a population of Macroptilium yellow spot virus (MaYSV) obtained from leguminous weeds in northeastern Brazil
ORFaTajima’s DFu and Li’s D*Fu and Li’s F*dN/dS
  1. aRep, replication-associated protein; Trap, trans-activating protein; Ren, replication enhancer protein; CP, coat protein.

  2. bns, not significant values at P < 0·10.

Rep0·8439 (ns)b0·6996 (ns)0·8319 (ns)0·7631
Trap−0·0477 (ns)−0·3255 (ns)−0·2892 (ns)0·4545
REn−0·0518 (ns)−0·3323 (ns)−0·2960 (ns)0·2132
CP−0·7991 (ns)−1·4288 (ns)−1·4366 (ns)0·0643

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The incidence and severity of diseases caused by begomoviruses has increased dramatically in many areas of the world, including Brazil, because of the explosion of B. tabaci populations (Morales, 2006). The efficient dissemination and high polyphagy of the B biotype of B. tabaci has enabled the transmission of indigenous begomoviruses to new cultivated hosts, and the emergence of novel recombinant variants arising from mixed infections (Ribeiro et al., 2007). The important role that weeds and wild plants have played as sources of begomoviruses for tomato and other important crops in Brazil is becoming increasingly clear. This study investigated the species diversity and genetic variability of begomoviruses infecting leguminous weeds in northeastern Brazil to determine the significance of these hosts as begomovirus reservoirs.

Six begomovirus species were found out of 19 DNA-A clones: an isolate of EuYMV obtained from M. atropurpureum, three BGMV isolates from M. lathyroides, and four new species, one of them infecting Ce. brasilianum, two infecting M. lathyroides and one infecting Cal. mucunoides, Canavalia sp. and M. lathyroides. This result indicates a high species diversity of begomoviruses infecting leguminous weeds in Brazil, similar to what has been observed for malvaceous and solanaceous weed species in the country (Jovel et al., 2004; Castillo-Urquiza et al., 2008; Paprotka et al., 2010b). Furthermore, the detection of five distinct viruses in samples of M. atropurpureum and M. lathyroides indicates that Macroptilium spp. may act as ‘mixing vessels’ in which recombinant viruses may arise. Macroptilium lathyroides has been reported as a host of distinct begomoviruses in Central America and the Caribbean (Idris et al., 2003), although it had been previously ruled out as an inoculum source for begomovirus epidemics in Jamaica (Roye et al., 1999).

Phylogenetic analyses based on DNA-A sequences of begomoviruses from the Americas showed that the four new species cluster with Brazilian viruses. However, the Brazilian begomoviruses do not collectively form a distinct monophyletic group in relation to other viruses from the Americas. MaYNV, MaYSV and CenYSV grouped with tomato-infecting begomoviruses from Brazil, while MaYVV and the three BGMV isolates clustered with BGMV isolates previously reported also from Brazil. Interestingly, EuYMV was placed in a group comprising mostly viruses from Mexico, Central and South America, but also including Sida yellow leaf curl virus (SiYLCV), ToCmMV and Abutilon Brazil virus (AbBV) which have been obtained from samples collected in Brazil (Castillo-Urquiza et al., 2008; Paprotka et al., 2010a). This continent-wide phylogeographical mixing of begomovirus species in South America is in fact reminiscent of that reported for African begomoviruses (Bull et al., 2006; Lefeuvre et al., 2007b). It is also noteworthy that while the initially characterized South American begomoviruses segregated into crop- and weed-infecting clades (Rojas et al., 2005), most ‘crop-infecting’ clades now contain an assortment of newly reported ‘weed-infecting’ viruses.

Accumulating evidence suggests that recombination is a common and important source of genetic diversity in Brazilian begomoviruses (Galvão et al., 2003; Inoue-Nagata et al., 2006; Ribeiro et al., 2007). Recombinant begomoviruses have been directly implicated in the emergence of new diseases and epidemics on crops in many countries (Pita et al., 2001; Monci et al., 2002; García-Andrés et al., 2006, 2007a,b; Lefeuvre et al., 2010). The analysis in the present study revealed that recombination is a common event among begomoviruses in leguminous weeds. Three recombination events were detected for MaYSV isolates, two of them (in the CP gene) having BGMV and MaYNV as the putative parents, and the other (in the Rep gene) having BlYSV as the putative parent. The close relationship between MaYSV and BlYSV was confirmed by phylogenetic analysis, in which these two viruses formed a group with 100% Bpp. Interestingly, BlYSV has been found, so far, only in the weed Blainvillea rhomboidea, from the family Asteraceae (Castillo-Urquiza et al., 2008). It remains to be demonstrated whether MaYSV and BlYSV have hosts in common.

The recombination events detected occurred primarily in the CP and Rep genes. However, one recombination breakpoint was found in the common region (CR) of MaYSV isolates BR:Bas1:09, BR:Mac1:10 and BR:Mac5:10 (Table S3). The CR is well characterized as a hot spot of recombination (Padidam et al., 1999). Although coding regions are generally less susceptible to recombination (Lefeuvre et al., 2007a), the begomovirus CP and Rep coding regions have also been demonstrated to contain recombination hot spots (García-Andrés et al., 2007b; Lefeuvre et al., 2007b).

The results of this study indicate that MaYSV is capable of infecting at least three weed species, M. lathyroides, Cal. mucunoides and Canavalia sp. MaYSV was detected in 12 out of 24 samples collected in three different states (Alagoas, Paraíba and Sergipe), and therefore seems to be the most common begomovirus in leguminous weeds in northeastern Brazil. However, further studies are needed to confirm this assumption.

Analysis of the MaYSV population demonstrated that its genetic variability is very high, with each isolate representing a single haplotype. This high variability is further demonstrated by high rates of nucleotide diversity, haplotype diversity and mutation. These values were considerably higher than those observed for two populations of tomato-infecting begomoviruses from southeastern Brazil (Castillo-Urquiza et al., 2010), and were similar to those observed for a BGMV population obtained from lima bean (P. lunatus) samples collected in Alagoas state (Ramos-Sobrinho et al., 2010). Therefore, it is tempting to conclude that begomoviruses infecting weed/wild hosts have a greater degree of genetic variability than viruses infecting crop species.

As with all viruses, the evolution of begomoviruses depends primarily on mutations. There is evidence that the rapid evolution of geminiviruses is, at least in part, driven by mutational processes acting specifically on ssDNA (Harkins et al., 2009). High mutation rates, similar to those observed for RNA viruses, have been estimated for the begomoviruses Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl virus (TYLCV) and East African cassava mosaic virus (EAMCV) and for the mastrevirus Maize streak virus (MSV) (Ge et al., 2007; Duffy & Holmes, 2008, 2009; Harkins et al., 2009). However, it has been reported that Brazilian BGMV isolates have an unusually low degree of genetic variability (Faria & Maxwell, 1999). This study was conducted before RCA greatly simplified the cloning of full-length begomovirus components and low-cost DNA sequencing became available. Therefore, the analysis was based on partial sequences, possibly underestimating the true genetic variability of the virus. Indeed, studies conducted with a BGMV population infecting lima bean showed that the variability within this species is high (Ramos-Sobrinho et al., 2010).

Neutrality tests were performed to assess whether there was evidence of selection or demographic forces acting on the MaYSV population. dN/dS values <1 were found for MaYSV, indicating that the MaYSV population may be under the influence of purifying selection or has undergone a recent expansion, so that the occurrence of mutations is not sufficient to fully explain its genetic variability. This also reinforces the possible influence of additional evolutionary forces (such as recombination) acting upon the population.

The findings of this study indicate that leguminous weeds such as M. lathyroides, M. atropurpureum, Canavalia sp. and Ce. brasilianum constitute important reservoirs of begomovirus species. Macroptilium spp. may also act as a mixing vessel that facilitates the emergence of novel viruses by recombination. This hypothesis is reinforced by the detection of recombination events in the MaYSV population. It is concluded that recombination as well as mutation is an important evolutionary process in the genetic diversification of the MaYSV population. Additional studies are necessary to demonstrate that weed species play an active role in begomovirus epidemics in crop plants, either by acting as primary inoculum sources or as a continuous source of novel viruses, which could disrupt management strategies based on the deployment of resistant varieties.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors wish to thank Enrique Moriones, Poliane Alfenas-Zerbini and Simone Ribeiro for critical review of the manuscript. This work was carried out under the framework of a Capes PROCAD-NF (no. 93-2008) collaborative project between UFAL, UFRPE and UFV, and was additionally funded by Fapemig grants CAG-666-08 and CAG-949-09 to FMZ. GPCU was the recipient of a Capes-PNPD postdoctoral fellowship.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Geographical map of the Brazilian northeastern states of Alagoas (AL), Paraíba (PB), Pernambuco (PE) and Sergipe (SE), indicating the locations where samples of leguminous weeds were collected

Figure S2. Neighbour-joining tree based on the complete DNA-A nucleotide sequences of begomoviruses from the Americas, including the viruses infecting leguminous weeds in northeastern Brazil. The four novel species detected in leguminous weeds (CenYSV, Centrosema yellow spot virus; MaYVV, Macroptilium yellow vein virus; MaYSV, Macroptilium yellow spot virus; and MaYNV, Macroptilium yellow net virus) are indicated in bold face. The 12 isolates belonging to MaYSV are underlined. Numbers at each branch indicate bootstrap confidence values (2000 replications).

Figure S3. Phylogenetic evidence for recombination among the DNA-A of begomoviruses from leguminous weeds in northeastern Brazil. Neighbour-net network analysis was performed using SplitsTree4. Formation of a reticular network rather than a bifurcated tree indicates the occurrence of recombination.

Figure S4. Phylogenetic evidence for recombination among the DNA-A of Macroptilium yellow spot virus (MaYSV) isolates. Neighbour-net network analysis was performed using SplitsTree4. Formation of a reticular network rather than a bifurcated tree indicates the occurrence of recombination.

Table S1. Characteristics of the genomic components (DNA-A and DNA-B) of begomovirus isolates corresponding to the four novel species detected in samples of leguminous weeds from northeastern Brazil.

Table S2. Percentage identities between the complete DNA-A nucleotide sequences of the six begomovirus species detected in leguminous weeds in four states of northeastern Brazil.

Table S3. Putative recombination events detected among isolates of Macroptilium yellow spot virus (MaYSV) infecting leguminous weeds in northeastern Brazil

Table S4. Putative recombination events detected among begomoviruses infecting leguminous weeds in northeastern Brazil.

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