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Virus Research 166 (2012) 13– 22

Contents lists available at SciVerse ScienceDirect

Virus Research

jo u r n al hom epa ge: www.elsev ier .com/ locate /v i rusres

olecular characterisation of dicot-infecting mastreviruses from Australia

ames Hadfielda, John E. Thomasb, Mark W. Schwinghamerc, Simona Krabergera, Daisy Staintona,nisha Dayarama, Judith N. Parryd, Daniel Pandee, Darren P. Martin f, Arvind Varsania,g,h,∗

School of Biological Sciences, University of Canterbury, Ilam, Christchurch 8140, New ZealandThe University of Queensland, Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation, Ecosciences Precinct, PO Box 46, Brisbane, QLD 4001, AustraliaDepartment of Employment, Economic Development and Innovation, 80 Meiers Road, Indooroopilly, Queensland 4068, AustraliaNSW Department of Primary Industries, Tamworth Agricultural Institute, 4 Marsden Park Road, Calala, NSW 2340, AustraliaDepartment of Botany and Horticulture, Maseno University, P.O. Box 333, Maseno, KenyaComputational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South AfricaElectron Microscope Unit, University of Cape Town, Rondebosch, 7701 Cape Town, South AfricaBiomolecular Interaction Centre, University of Canterbury, Ilam, Christchurch 8140, New Zealand

r t i c l e i n f o

rticle history:eceived 21 December 2011eceived in revised form 16 February 2012ccepted 20 February 2012vailable online 3 March 2012

eywords:

a b s t r a c t

Monocotyledonous and dicotyledonous plant infecting mastreviruses threaten various agricultural sys-tems throughout Africa, Eurasia and Australasia. In Australia three distinct mastrevirus species are knownto infect dicotyledonous hosts such as chickpea, bean and tobacco. Amongst 34 new “dicot-infecting”mastrevirus full genome sequences obtained from these hosts we discovered one new species, four newstrains, and various variants of previously described mastrevirus species. Besides providing additionalsupport for the hypothesis that evolutionary processes operating during dicot-infecting mastrevirus

icot-infecting mastrevirusesecombinationeminivirusustraliahickpea

evolution (such as patterns of pervasive homologous and non-homologous recombination, and strongpurifying selection acting on all genes) have mostly mirrored those found in their monocot-infectingcounterparts, we find that the Australian dicot-infecting viruses display patterns of phylogeographicclustering reminiscent of those displayed by monocot infecting mastrevirus species such as Panicumstreak virus and Maize streak virus.

eanobacco

. Introduction

In Australia chickpea (Cicer arietinum) and bean (Phaseolus vul-aris) are important crops worth more than AUD 250 million aear (Anonymous, 2011a,b). Amongst the most important biotichreats to Australian bean and chickpea cultivation are virusesn the Mastrevirus genus of the family Geminiviridae (Ballantynet al., 1969; Schwinghamer et al., 2010). Such dicotyledonous-plantnfecting mastreviruses also cause major losses in tobacco (Nico-iana tabacum, family Solanaceae) (Hill and Allan, 1942).

Mastreviruses are leafhopper-transmitted, single stranded DNAiruses with 2.5–2.7 kb long circular genomes. The genus containsoth species that infect grasses (Poaceae including small grainsnd maize) and species that infect dicotyledonous plants in Africa,

urope, western and southern-central Asia, and Australia (Brownt al., 2011). Mastreviruses that infect dicots include the recog-ised species Tobacco yellow dwarf virus (TYDV) (Morris et al., 1992)

∗ Corresponding author at: School of Biological Sciences, University of Canterbury,rivate Bag 4800, Christchurch 8140, New Zealand. Tel.: +64 3 366 7001x4667;ax: +64 3 364 2590.

E-mail address: arvind.varsani@canterbury.ac.nz (A. Varsani).

168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2012.02.024

© 2012 Elsevier B.V. All rights reserved.

from Australia and Bean yellow dwarf virus (BeYDV) (Liu et al.,1999) from southern Africa and Pakistan and proposed specieschickpea chlorosis virus (CpCV-A, CpCV-B) and chickpea redleafvirus (CpRLV) from Australia (Thomas et al., 2010). Other tenta-tive species such as chickpea chlorotic dwarf virus (CpCDV) fromnorthern Africa and the Indian subcontinent (Horn et al., 1993),chickpea chlorotic dwarf Pakistan virus (CpCDPKV; Nahid et al.,2008), chickpea chlorotic dwarf Syria virus (CpCDSV; Mumtaz et al.,2011), and chickpea chlorotic dwarf Sudan virus (CpCDSDV; Aliet al., 2004) probably represent strains of a single species. Whereverhost ranges have been examined, mastreviruses in this group havebeen found to be capable of infecting a diverse range of dicotyle-donous plant families (although in many cases the identity of theviruses has not been confirmed at the molecular level; Thomas andBowyer, 1984; Trebicki et al., 2010). CpCDV infects sugar beet (Betavulgaris, family Amaranthaceae) naturally in Iran (Farzadfar et al.,2008) and TYDV infects species in at least seven plant families,and occurs naturally in a number of these, including tobacco, beanand chickpea (Thomas and Bowyer, 1984; Trebicki et al., 2010).

Infection is lethal in susceptible bean cultivars (Ballantyne et al.,1969; Rybicki and Pietersen, 1999) and early-infected chickpea(Horn et al., 1995) but is more often sub-lethal to symptomless.Sub-lethal but often damaging symptoms in dicotyledonous hosts

1 Resea

t(

m2cmNad(

AscpsiAttptptem

2

2

iih(eccf

2

emcHgeaoTA((cait

2

s

4 J. Hadfield et al. / Virus

ypically include stunting and chlorosis or reddening of foliageSchwinghamer et al., 2010).

Relative to monocot-infecting mastreviruses the dicot-infectingastreviruses comprise a biologically, serological (Brown et al.,

011) and phylogenetically distinct group. Although Australia isurrently the source of the most diverse array of dicot-infectingastreviruses (Thomas et al., 2010), a recent survey in theew South Wales and Queensland states of that country, found

low incidence (<5%) of mastrevirus infections amongst ran-omly sampled cultivated legumes and their associated weedsSchwinghamer et al., 2010).

Previous analysis of dicot-infecting mastreviruses from easternustralia (TYDV, CpCV-A, CpCV-B, CpRLV) revealed greater diver-ity than that found in Africa and Asia (i.e. for BeYDV and CpCDV)ombined (Thomas et al., 2010), thereby suggesting that Australia isotentially the global hotspot of dicot-infecting mastrevirus diver-ity. This indicated that further mastrevirus diversity could existn chickpea and possibly other potential cultivated host species inustralia. Here we determine and analyse 34 new complete mas-

revirus genome sequences from twenty chickpea, five bean, andwo tobacco plants. In addition to contributing sequences to thereviously characterised virus species we identify three poten-ially novel CpCV strains, a potentially novel TYDV strain, and aotentially novel chickpea-infecting mastrevirus species. This addi-ional sampling enables increased resolution in analyses of thevolutionary processes that are operational during dicot-infectingastrevirus evolution.

. Materials and methods

.1. Sampling

Diced/dried leaf samples and frozen leaf extracts represent-ng individual chickpea, bean, or tobacco plants were identified asnfected with mastreviruses in earlier studies by M.W. Schwing-amer J.E. Thomas, J.N. Parry and collaborators in New South WalesNSW), Victoria (Vic) and Western Australia (WA) (Schwinghamert al., 2010). Thirty-three samples (see Table 1 for details) wereollected mainly from eastern Australia with the exception of oneollected in western Australia. These samples included twenty-sixrom chickpea, five from bean and two from tobacco.

.2. Genome cloning

Total genomic DNA was isolated using the ISOLATE plant DNAxtraction kit (Bioline) as per the manufacturer’s instructions. Oneicrolitre of extracted DNA was used for non-specific rolling-

ircle amplification using Phi29 DNA polymerase (TempliPhiTM, GEealthcare, USA) as described previously for the amplification ofeminivirus genomes (Shepherd et al., 2008). Concatemers wereither cut with the restriction enzymes BamHI, XmnI, SacI or PstInd ligated directly into similarly linearised pGEM3Zf (Promega)r were amplified using back-to-back primers (forward: 5′—GANTG GTC CGC AGA TGT AGA G—3′ and reverse: 5′—GTA CCG GWAGA CMW CYT GGG C—3′) using a hi-fidelity DNA polymerase

AccuzymeTM, Bioline, USA) (see Table 1) and ligated to pJET1.2Fermentas, USA). The primers were designed back-to-back in aonserved Rep region of the genomes (based on an alignment of allvailable dicot-infecting mastrevirus genome sequences availablen Genbank). Plasmids containing viral genome fragments werehen sequenced by primer walking at Macrogen Inc. (South Korea).

.3. General sequence analyses and manipulations

Viral genome assemblies were carried out using DNAMAN (ver-ion 5.2.9; Lynnon Biosoft). ORFs were extracted and spliced in

rch 166 (2012) 13– 22

silico based on detectable homology with previously publisheddicot-infecting mastreviruses using MEGA (version 5) (Tamuraet al., 2011). Full genome and ORF alignments were generally per-formed using ClustalW (Larkin et al., 2007) with checking by eye,but the entire full and partial genome datasets of other mastrevirusspecies such as maize streak virus (MSV), Panicum streak virus(PanSV) and Wheat dwarf virus (WDV) were aligned using MUS-CLE (Edgar, 2004). Sequence logos were produced using WebLogo(Crooks et al., 2004).

2.4. Recombination analyses

RDP4 (version 4.13) (Martin et al., 2010) was used for recom-bination analysis with recombination signals being detected usingthe RDP (Martin and Rybicki, 2000), BOOTSCAN (Martin et al., 2005),MAXCHI (Smith, 1992), CHIMERA (Posada and Crandall, 2001),SiScan (Gibbs et al., 2000) and 3SEQ (Boni et al., 2007), meth-ods. Maximum likelihood phylogenetic trees were constructedusing PhyML (Guindon and Gascuel, 2003) using the GTR + G4model (chosen by ModelTest as implemented in RDP4 (Posada andCrandall, 1998) for nucleotide alignments and the LG model foramino-acid alignments.

2.5. Selection analyses

Ratios of normalised synonymous (dS) and non-synonymous(dN) substitution rates were estimated from codon alignedmovement protein (MP), coat protein (CP) and replicationassociated protein (Rep) gene nucleotide sequences using therecombination-aware version of the SLAC method (Pond andFrost, 2005; Pond et al., 2006) implemented in DATAMONKEY(http://www.datamonkey.org/; Delport et al., 2010). Besides thedicot-infecting mastrevirus datasets we also analysed analogousMP, CP and Rep datasets assembled from full genome sequencealignments of various monocot-infecting mastreviruses (1) 439MSV isolates, (2) 39 PanSV isolates and (3) 78 European dwarfvirus isolates (EDV; including WDV, barley dwarf virus and oatdwarf virus; all alignments are available on request from theauthors). Crucially, despite the fact that the EDV and the dicot-infecting mastrevirus datasets contain sequences from multiplespecies whereas the MSV and PanSV datasets each containedsequences from only single species, all of these datasets actu-ally contained similar degrees of sequence diversity and thereforelikely contain signals of natural selection operating over similartimescales.

3. Results and discussion

3.1. Classification of 34 new Australian dicot-infectingmastrevirus genome sequences

We isolated, cloned and sequenced 34 full length mastrevirusgenomes from 28 plants (chickpea, n = 21; bean, n = 5; tobacco,n = 2). We were unable to idolate full genomes from 5 chickpeaplants (samples 29–33; Table 1). Although we were unable toisolate full genomes from five additional chickpea samples, we nev-ertheless managed to isolate a number of sub-full length genomicDNA molecules (hereafter referred to as subgenomic molecules)from these samples. The 34 newly sequenced genomes and 13 pre-viously determined dicot-infecting mastrevirus genomes obtainedfrom GenBank were aligned using MUSCLE (Edgar, 2004) andMEGA5 (Tamura et al., 2011) and used to determine a genetic

distance matrix (p-distances with pairwise deletion of gaps – amore conservative measure than when using gaps as a fifth charac-ter). The distribution of these distance values (determined froma total of 861 pairwise comparisons) indicated that the current

J. H

adfield et

al. /

Virus

Research

166 (2012) 13– 2215

Table 1Information on nucleotide sequences of dicot-infecting mastreviruses determined in this study and other studies. The 47 sequences above the line were complete genome sequences and other 13 below the line were partialsequences that also occurred in vivo. The use of high-fidelity PCR or restriction enzyme in genome isolation (see Section 2) is indicated. DAR denotes NSW Plant Pathology Herbarium, Orange, NSW; BRIP denotes QueenslandPlant Pathology Herbarium, Ecosciences Precinct, Dutton Park, Queensland.

Sample # Field specimen ID Voucher specimen Host Sampling year Location Digest/PCR Isolate GenBank accession

1 196 Tobacco 1986 Myrtleford, VIC, Australia BamHI TYDV-A [AU:196:1986] JN989440 Fulllengths2 2562 Bean 2010 Yanco, NSW, Australia BamHI TYDV-A [AU:2562:2010] JN989442

3 2564 Bean 2010 Yanco, NSW, Australia BamHI TYDV-A [AU:2564:2010] JN9894434 48 Tobacco 1985 Myrtleford, VIC, Australia BamHI TYDV-A [AU:48:1985] JN9894455 2561 Bean 2010 Yanco, NSW, Australia PCR TYDV-A [AU:2561:2010] JN9894416 3492M Chickpea 2002 Narromine. NSW, Australia PCR TYDV-A [AU:3492M:2002] JN989444

TYDV-C [AU:3492M:2002] JN9894467 3768F DAR 81662 Chickpea 2007 Tomingley/Peak Hill, NSW, Australia SmaI CpCV-D [AU:3768F:2003] JN9894238 2008 Bean 2007 Bairnsdale, VIC, Australia PCR CpCV-D [AU:2008:2007] JN9894189 3494I DAR 81660 Chickpea 2002 Warren, NSW, Australia PCR CpCV-D [AU:3494I:2002] JN98942210 2614 Chickpea 2010 Clermont, QLD, Australia PCR CpCV-D [AU:2614:2010] JN989420

CpCV-D [AU:2614a:2010] JN98942111 2612 Chickpea 2010 Clermont, QLD, Australia PCR CpCV-D [AU:2612:2010] JN98941912 3459G Chickpea 2002 Narrabri, NSW, Australia PCR CpCV-E [AU:3459G:2002] JN98942513 3498E Chickpea 2002 Breeza, NSW, Australia PCR CpCV-E [AU:3498Ea:2002] JN989435

XmnI CpCV-E [AU:3498Eb:2002] JN98943614 84 Bean 1984 Pemberton, WA, Australia XmnI CpCV-E [AU:84:1984] JN98943815 3495M DAR 76860 Chickpea 2002 Warren, NSW, Australia PCR CpCV-E [AU:3495Mb:2002] JN989433

XmnI CpCV-E [AU: 3495Ma:2002] JN98943216 3487P Chickpea 2002 Gilgandra, NSW, Australia PCR CpCV-E [AU:3487P:2002] JN98942817 3495D Chickpea 2002 Warren, NSW, Australia PCR CpCV-E [AU:3495D:2002] JN989430

CpCV-E [AU:3495Da:2002] JN98943118 3489A Chickpea 2002 Gilgandra, NSW, Australia SacI CpCV-E [AU:3489A:2002] JN98942919 3498K Chickpea 2002 Breeza, NSW, Australia XmnI CpCV-E [AU:3498K:2002] JN98943720 3458H Chickpea 2002 Narrabri, NSW, Australia PCR CpCV-E [AU:3458H:2002] JN98942421 3487H Chickpea 2002 Gilgandra, NSW, Australia PCR CpCV-E [AU:3487H:2002] JN98942722 3460B DAR 76859 Chickpea 2002 Narrabri, NSW, Australia XmnI CpCV-E [AU:3460B:2002] JN98942623 3498A Chickpea 2002 Breeza, NSW, Australia XmnI CpCV-E [AU:3498A:2002] JN98943424 2683A Chickpea 2002 Dalby, QLD, Australia XmnI CpCV-A [AU:2683A:2002] JN98941325 3494K Chickpea 2002 Warren, NSW, Australia PCR CpCV-A [AU:3494K:2002] JN98941526 3459F Chickpea 2002 Narrabri, NSW, Australia PCR CpCV-A [AU:3459F:2002] JN98941427 3494I DAR 81660 Chickpea 2002 Warren, NSW, Australia PCR CpCV-C [AU:3494Ib:2002] JN989417

CpCV-C [AU:3494Ia:2002] JN98941628 3489B DAR 81659 Chickpea 2002 Gilgandra, NSW, Australia SacI CpYV [AU:3489B:2002] JN989439

– Tobacco 1992 Myrtleford, VIC, Australia – TYDV-A [AU:1992] M81103Qld 22 BRIP 52878a Chickpea 2003 Emerald, QLD, Australia – CpRLV [AU:Qld22:2003] GU2565323455C DAR 76857 Chickpea 2002 Croppa Creek, NSW, Australia – CpCV-A [AU:3455C:2002] GU256530Qld 21 BRIP 52877a Chickpea 2002 Emerald, QLD, Australia – CpCV-B [AU:Qld21:2003] GU256531CCDV6 Chickpea 2005 Faisalabad, Pakistan – CpCDPKV [PK:ccdv6:2005] AM849097CCDV8 Chickpea 2007 Layyah, Pakistan – CpCDPKV [PK:ccdv8:2007] AM900416CCDV3 Chickpea 2007 Layyah, Pakistan – CpCDPKV [PK:ccdv3:2007] AM850136CCDEV14 Chickpea 2005 Faisalabad – BeYDV [PK:ccdv14:2005] AM849096SA Bean South Africa – BeYDV [SA] Y11023SA-mild Bean South Africa – BeYDV [SA:mild] DQ458791Nhe5 Chickpea 1997 Abu Haraz, Sudan – CpCDSDV [SD:Nhe5:1997] AM933134Eco18 Chickpea 1997 Abu Haraz, Sudan – CpCDSDV [SD:Eco18:1997] AM933135

16 J. Hadfield et al. / Virus Resea

Tabl

e

1

(Con

tinu

ed)

Sam

ple

#

Fiel

d

spec

imen

ID

Vou

cher

spec

imen

Hos

t

Sam

pli

ng

year

Loca

tion

Dig

est/

PCR

Isol

ate

Gen

Ban

k

acce

ssio

n

Syr2

Ch

ickp

ea

2008

Syri

a

–

Cp

CD

SV

[SY

R:S

YR

2:20

08]

FR68

7959

2034

58H

Ch

ickp

ea20

02N

arra

bri,

NSW

, Au

stra

lia

PCR

3458

H

(Cp

CV

-E-l

ike)

–Su

b-ge

nom

icm

olec

ule

s29

3487

M

Ch

ickp

ea

2002

Gil

gan

dra

, NSW

, Au

stra

lia

PCR

3487

Ma

(Cp

CV

-E-l

ike)

–34

87M

b

1.5

(Cp

CV

-E-l

ike)

1634

87P

Ch

ickp

ea

2002

Gil

gan

dra

, NSW

, Au

stra

lia

PCR

3487

P

(Cp

CV

-E-l

ike)

–30

3495

IC

hic

kpea

2002

War

ren

, NSW

, Au

stra

lia

PCR

3495

Ia

1.5

(Cp

CV

-E-l

ike)

–34

95Ib

(Cp

CV

-E-l

ike)

3137

58E

DA

R

8166

1C

hic

kpea

2003

Du

bbo,

NSW

, Au

stra

lia

PstI

3758

E

(Cp

CV

-D-l

ike)

–32

3489

C

Ch

ickp

ea

2002

Gil

gan

dra

, NSW

, Au

stra

lia

PCR

3489

C

(Cp

CV

-E-l

ike)

–33

3489

HPC

R34

89H

a(C

pC

V-E

-lik

e)–

3489

Hb

(Cp

CV

-E-l

ike)

3489

H

c(C

pC

V-E

-lik

e)27

3494

I

DA

R

8166

0

Ch

ickp

ea

2002

War

ren

, NSW

, Au

stra

lia

PCR

3494

I a(C

pC

V-C

-lik

e)

–34

94I b

(Cp

CV

-C-l

ike)

rch 166 (2012) 13– 22

International Committee on the Taxonomy of Viruses (ICTV; Brownet al., 2011) <75% pairwise sequence identity criterion for speciesdemarcation and the tentative push towards a global geminivirus<89% pairwise identity criterion for species demarcation may needto be reviewed as it is possibly less appropriate than an 84%dicot-infecting matsrevirus species demarcation criterion (Fig. 1).Specifically, four main clusters of pairwise distances are evidentbetween 63–70%, 74–82% and 85–87 and 93–100%. Based on theseclusters it may be reasonable to have isolates sharing > 84% pair-wise identity belonging to the same species, isolates sharing >95%pairwise identity belonging the same strain and isolates sharing>95% pairwise identity belonging to the same variant grouping. Itshould be noted, however, that the pairwise distance comparisonspresented in Fig. 1 are possibly biased because the dataset under-lying the presented analysis contained mostly Australian viruses.It is therefore possible a similar analysis containing more iso-lates from Asia, Africa and the Middle East could be less biasedand help provide some resolution to our tentative classificationscheme.

If the 84% species demarcation criterion is applied to the dicot-infecting mastreviruses, then the Australian viruses would be splitinto eight species (isolates of TYDV, CpRLV, CpYV, CpCV-A, CpCv-B,CpCVC, CpCV-D and CpCV-E would represent distinct species). Con-versely, all four previously published genomes from Pakistan, Africaand Syria including the various BeYDV and CpCDV variants wouldbe grouped into a single species. Despite our misgivings regard-ing the current classification schemes (multiple schemes are usedby different authors), for the purpose of this study we have nev-ertheless adhered to the current ICTV sanctioned 75% mastrevirusspecies demarcation criterion.

Based on phylogenetic (Fig. 2) and pairwise genetic distancefrequency analyses (Fig. 1) and the ICTV’s current 75% mastre-virus species demarcation criterion, we tentatively defined one newspecies, provisionally named Chickpea yellows virus (CpYV), whichsits on a divergent branch within the tree and has a genome <69.6%similar to those of any other currently classified dicot-infectingmastrevirus species (Fig. 3). Given that isolates sharing between75 and 95% pairwise identity belong to the same strain we iden-tified three new strains of CpCV (CpCV-C, D, and E) and one newstrain of TYDV (TYDV-C) distinct from both the first full-lengthTYDV sequence to be determined (Morris et al., 1992) which wehere call TYDV-A and another partially sequenced TYDV-B (Thomaset al., 2010). Of the three new CpCV strains, two (CpCV-D and -C)could, along with the previously characterised CpCV-B strain, alsopotentially be classified as new species as they share <78% similar-ity (conservatively calculated using pairwise deletion of gaps) withall other dicot-infecting mastreviruses.

3.2. Motifs in Rep

For all genome sequences obtained in this work, as for othermastreviruses, there is a highly conserved sequence domain nearthe start of the predicted Rep protein. An iteron-related domain(IRD) signal, thought to bind to iterated sequences (or iterons) nearthe virion strand origin of replication and found in all geminiviruses(Arguello-Astorga and Ruiz-Medrano, 2001), is highly conservedhaving the sequence logo AFRLQTKYVFLTYP(H/R)C (SupplementaryFig. 1A). The geminivirus Rep sequence (GRS) motif, shown to beessential for the nicking of ssDNA during the initiation of rollingcircle replication (Nash et al., 2011), is mostly present but lackscentral residues (Supplementary Fig. 1). These central residues,thought to be related to dicotyledonous host adaptation (Nash

et al., 2011), are apparently not required by all dicot-infectinggeminiviruses. All rolling circle replication associated motifs, theretinoblastoma protein binding domain, and dNTP binding motifscharacteristic of mastrevirus Rep sequences were identified within

J. Hadfield et al. / Virus Research 166 (2012) 13– 22 17

Percentage pairwise identity

Pro

po

rtio

n o

f p

air

wsie

id

en

titi

es

Species

Strains

Variants

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

10099989796959493929190898887868584838281807978777675747372717069686766656463626160

F icot-iw CLE fu

tF

3s

iefwpbstdplMC

mb(gsaM

ig. 1. Distribution of percentage pairwise genetic similarity between individual dere calculated using p-distances (with pairwise deletion of gaps) based on a MUS

he Rep sequences of the newly sequenced viruses (Supplementaryig. 1).

.3. Detection and characterisation of sub-genome length viralequences in dicotyledonous plants

In addition to the 34 full genomes that we characterised, wesolated thirteen sub-genome length sequences from eight differ-nt chickpea plants (Fig. 4). These so-called sub-genomics rangingrom 306 to 2036 nt long with an average length of 1300 nt. Tenere CpCV-E-like, two CpCV-C-like, and one CpCV-D-like based onhylogenetic analysis (Supplementary Fig. 2B). In three cases whereoth subgenomic and full-length sequences were isolated from theame plant the subgenomic sequences were nearly identical toheir associated full-length sequences. These potentially non-viableefective sub-genomics appeared to have recombination break-oints that were non-randomly distributed and significantly more

ikely to occur in the two intergenic regions (�2 p-value 0.000122).ost lacked genome segments in virion sense ORF regions (MP and

P).Despite the fact that we likely preferentially amplified

olecules with intact Rep regions due to the location of the primerinding sites we used during genome amplification and cloningFig. 4), the pattern of sequence deletion seen in the CpCV sub-

enomics is strikingly similar to that noted previously in MSVub-genomics seen both during controlled laboratory infectionsnd in field isolated samples (van der Walt et al., 2009). As with theSV sub-genomics, the breakpoint distributions displayed by the

nfecting mastreviruses, rounded to the nearest whole number. Pairwise identitiesll genome alignment.

CpCV sub-genomics are very similar to homologous recombinationbreakpoint distributions seen in field isolated mastreviruses with amajor recombination hotspot immediately 5′ of the primer bindingsite at the origin of complementary strand replication (Garcia-Andres et al., 2007; Lefeuvre et al., 2007; Schnippenkoetter et al.,2001; Stenger et al., 1991; Varsani et al., 2008).

3.4. Recombination patterns in the dicot-infecting mastreviruses

Genetic recombination has played a prominent role in gemi-nivirus evolution (Harkins et al., 2009a,b; Lefeuvre et al., 2007;Martin et al., 2011b; Padidam et al., 1999; Varsani et al., 2008).Only recently with the determination of sufficient dicot-infectingmastrevirus full genome sequences, has it become possible toinfer that recombination patterns between dicot-infecting mas-treviruses may precisely mirror those found in monocot-infectingmastreviruses (Martin et al., 2011a,b). Here in an analysis ofconsiderably more sequences we provide further evidence insupport of this possibility (Fig. 2). A total of 12 recombinationevents were detected amongst the dicot mastreviruses (Fig. 2).The new sequences reported here refine the previous recom-bination maps and, in three cases at least, redefine sequencespreviously identified as parental as being recombinants and viceversa. Specifically, TYDV is likely to be a recombinant of CpCV-

D (Australia) and a virus distantly related to CpCDV (Pakistan,Sudan and Syria; event 1 in Fig. 2). Similarly, the new strainCpCV-C is a likely recombinant of CpCV-A and a sequence resem-bling an ancestor of TYDV (event 7 in Fig. 2). Whilst CpCV-A

18 J. Hadfield et al. / Virus Research 166 (2012) 13– 22

SYR

1

1 2

3 4 10

65

77

89

11

12

MP

CP Rep

TYDV

CpCV-D

CpCDPKV, BeYD V, CpCDSDV & CpCDSV

TYDV ancestral

CpCDPKV, BeYD V, CpCDSDV & CpCDSV

ancestral

CpCV-E

CpCV-E ancestral

CpCV-D ancestral

unknown

4 10

7

RepA

CpCDPKV3 [PK:ccdv3:2007]BeYDV1 [PK:ccdv14:2005]

CpCDSV1 [SYR:SYR2:2008]

CpCDSDV1 [SD:Nhe5:1997]

BeYDV1 [SA]

BeYDV2 [SA:mild]

CpCDSDV1 [SD:Eco18:1997]

CpCDPKV2 [PK:ccdv8:2007]

CpCDPKV1 [PK:ccdv6:2005]

0.07

TYDV-A1 [AU:1992]

CpCV-A3 [AU:2683A:2002]

CpCV-E1 [AU:3460B:2002]

CpCV-A1 [AU:3459F:2002]

CpCV-E11 [AU:3489A:2002]

CpCV-E1 [AU:3498Ea:2002]

CpCV-E1 [AU:3495Da:2002]

CpCV-A1 [AU:3455C:2002]

CpCV-D1 [AU:2008:2007]

CpCV-E1 [AU:3487P:2002]

CpCV-E1 [AU:3495Ma:2002]

CpCV-C1 [AU:3494I:2002]

CpCV-EI [AU:3498K:2002]

CpCV-EI [AU:3498A:2002]

CpCV-C1 [AU:3494Ia:2002]

TYDV-C1 [AU:3492Mb:2002]

CpCV-D1 [AU:2614a:2010]

TYDV-A5 [AU:2561:2010]

TYDV-A2 [AU:2562:2010]

CpCV-E1 [AU:3495Mb:2002]

TYDV-A4 [AU:48:1985]

CpCV-D1 [AU:3494I:2002]

TYDV-A2 [AU:196:1986]

CpCV-B1 [AU:Qld21:2003]

CpYV1 [AU:3489b:2002]

CpCV-E1 [AU:3498Eb:2002]

CpCV-E1 [AU:84:1984]

CpCV-E1 [AU:3459G:2002]

CpCV-E1 [AU:3487H:2002]

CpCV-D1 [AU:2612:2010]

TYDV-A6 [AU:3492Ma:2002]

CpCV-A2 [AU:3494K:2002]

CpCV-E1 [AU:3458H:2002]

CpRLV1 [AU:Qld22:2003]

CpCV-D1 [AU:2614b:2010]

TYDV-A3 [AU:2564:2010]

CpCV-E1 [AU:3495Db:2002]

CpCV-D1[AU:3768F:2003]

Event

123456789

101112

BreakpointsRecombinant

Sequences used to

infer major parent

Sequences used to

infer minor parent Begin En d Methods p-Value

GR37121131VDCpClartsecnanwonknuVDYT BMCS T 2.45e-15TYDV-C TYDV-A CpCDV 1787 2023 RGB MCT 8.15e-11CpCV-A3 GR322921VDYTA-VCpC BMC S 5.90e-14

R17419511VDYTE-VCpCA-VCpC GBMCS T 1.16e-49CpCV-B CpCV-A unknow n 82 270 MCS 1.63e-05CpCV-B CpCV-E CpCV-C 1489 2468 MC S 7.80e-10CpCV-C ancestral TYDV CpCV-A 266 4 46 1 RBMC S 4.69e-11

BR29120421nwonknuA-VCpClartsecnaVRpC MCS 1.78e-09CpMV ancestral CpCV-D unknown 125 5 261 3 RBM CST 1.17e-07CpCV-A TVDV / CpCV-E CpCDV 1429 1465 GMT 7.34e-05

R481011VMpCVDCpCVDYeB GM 1.56e-04TYDV-A unknow n CpCV-D 176 1 183 1 RGBMC S 2.32e-05

BA

C

Fig. 2. (A) Maximum likelihood phylogenetic tree of Australian dicot-infecting mastrevirus complete genomes with the monocot-infecting mastrevirus, Oat dwarf virus,used as an outlier (not shown). Red text indicates genomes reported in this study. Closed circles indicate branches supported by greater than 90% of 1000 bootstrap replicateswhilst open circles represent branches supported by less than 90% but greater than 60% of bootstrap replicates. Branches with less than 60% bootstrap support have beenc recomd the red Q meti to the

sto3ttin2

migCh(r

ollapsed. See Table 1 for accession numbers. (B) Cartoon representation of detectedifferent lineages and event numbers correspond to those in panel C. (C) Details of

etection by the RDP, GENCONV, BOOTSCAN, MAXCHI, CHIMERA, SISCAN and 3SEnterpretation of the references to color in this figure caption, the reader is referred

eems to be a recombinant of CpCV-E and TYDV, the two geno-ypes still fall into separate clades when the recombinant sectionsf their sequences are removed (Supplementary Fig. 2A; event

& 4 in Fig. 2). No significant differences were observed inhe branching order of the maximum likelihood phylogeneticrees inferred from the full genome sequences and similar treesnferred using sequences from which the identified recombi-ant regions had been removed (Fig. 2 and Supplementary Fig.A).

The breakpoint distributions detected here for dicot-infectingastreviruses are very similar those seen previously in monocot-

nfecting mastreviruses. The complementary-sense ORFs of othereminiviruses (C1 and C2 in the case of mastreviruses and C1, C2,

3 and C4 in the case of the other geminivirus genera) exhibitigher basal recombination rates than their virion-sense ORFsOwor et al., 2007). Consistent with this view, we detected moreecombination breakpoints in the complementary sense genes than

bination events amongst the dicot-infecting mastreviruses. Colours correspond tocombination events detected using RDP4. R, G, B, M, C, S, T indicate recombinationhods, respectively, with the p-value shown for the method indicated in bold. (For

web version of the article.)

we did in the virion sense genes (seven vs. four). Also, consis-tent with recombination patterns seen in single stranded DNAviruses in general (Lefeuvre et al., 2009), we detected more break-points within the intergenic regions than within the coding regions.As with monocot-infecting mastreviruses (Martin et al., 2011b;Monjane et al., 2011; van der Walt et al., 2009; Varsani et al.,2009), we found the short intergenic region (SIR) to be a partic-ularly recombination prone region of dicot-infecting mastrevirusgenomes.

Recombination between the Australian dicot-infecting mastre-viruses is also clearly evident. Recombination with a non-Australianvirus may have occurred for TYDV, the Rep region of which is relateddistantly to the West Asian/African dicot-infecting mastreviruses

(event 1; Fig. 2). However, the geographical source of the ances-tral Rep sequence, and hence TYDV, will remain uncertain untilsequences more closely resembling its parental viruses are sampledand characterised.

J. Hadfield et al. / Virus Research 166 (2012) 13– 22 19

Fig. 3. Two-dimensional representation of pairwise genome-wide nucleotide sequence identities calculated with pairwise deletion of gaps, scale represents pairwise identity).New sequences are highlighted in red. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)

PCPM RepRepA

F/R

3458H (CpCV-E-like)3487Ma (CpCV-E-like)3497Mb (CpCV-E-like)

3487P (CpCV-E-like)3495Ia (CpCV-E-like)3495Ib (CpCV-E-like)3758E (CpCV-D-like)3489C (CpCV-E-like)

3487Ha (CpCV-E-like)3487Hb (CpCV-E-like)3487Hc (CpCV-E-like)3494Ia (CpCV-C-like)3494Ib (CpCV-C-like)

Fig. 4. Sub-genomic molecules characterised in this study. Schematic representation of the partial, circular, sub-genomic molecules when aligned (red) shown against a fullgenome scaffold (blue). The vertical black line represents the site of back-to-back primers used to isolate sub-genomic molecules and the hashed section of the Rep generepresents the intron. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)

20 J. Hadfield et al. / Virus Research 166 (2012) 13– 22

CpCDPKV [PK:ccdv3:2007]BeYDV [PK:ccdv14:2005]

CpCDSV [SYR:SYR2:2008]

CpCDSDV [SD:Nhe5:1997]

BeYDV [SA]BeYDV [SA:mild]

CpCDSDV [SD:Eco18:1997]

CpCDPKV [PK:ccdv8:2007]CpCDPKV [PK:ccdv6:2005]

TYDV-A [AU:1992]

CpCV-A [AU:2683A:2002]

CpCV-E [AU:3460B:2002]

CpCV-A [AU:3459F:2002]

CpCV-E [AU:3489A:2002]

CpCV-E [AU:3498Ea:2002]

CpCV-E [AU:3495Da:2002]

CpCV-A [AU:3455C:2002]

CpCV-D [AU:2008:2007]

CpCV-E [AU:3487P:2002]

CpCV-E [AU:349Ma:2002]

CpCV-C [AU:3494Ib:2002]

CpCV-E [AU:3498K:2002]

CpCV-E [AU:3498A:2002]

CpCV-C [AU:3494Ia:2002]

TYDV-C [AU:3492Mb:2002]

CpCV-D [AU:2614a:2010]

TYDV-A [AU:2561:2010]

TYDV-A [AU:2562:2010]

CpCV-E [AU:3495Mb:2002]

TYDV-A [AU:48:1985]

CpCV-D [AU:3494I:2002]

TYDV-A [AU:196:1986]

CpCV-B [AU:Qld21:2003]

CpYV [AU:3489B:2002]

CpCV-E [AU:3498Eb:2002]

CpCV-E [AU:84:1984]

CpCV-E [AU:3459G:2002]

CpCV-E [AU:3487H:2002]

CpCV-D [AU:2612:2010]

TYDV-A [AU:3492Ma:2002]

CpCV-A [AU:3494K:2002]

CpCV-E [AU:3458H:2002]

CpRLV [AU:Qld22:2003]

CpCV-D [AU:2614b:2010]

TYDV-A [AU:2564:2010]

CpCV-E [AU:3495Db:2002]

CpCV-D [AU:3768F:2003]

CpCDPKV [PK:ccdv3:2007]BeYDV [PK:ccdv14:2005]

CpCDSV [SYR:SYR2:2008]CpCDSDV [SD:Nhe5:1997]

BeYDV [SA]BeYDV [SA:mild]

CpCDSDV [SD:Eco18:1997]

CpCDPKV [PK:ccdv8:2007]CpCDPKV [PK:ccdv6:2005]

TYDV-A [AU:1992]

CpCV-A [AU:2683A:2002]

CpCV-E [AU:3460B:2002]

CpCV-A [AU:3459F:2002]

CpCV-E [AU:3489A:2002]

CpCV-E [AU:3498Ea:2002]

CpCV-E [AU:3495Da:2002]

CpCV-A [AU:3455C:2002]

CpCV-D [AU:2008:2007]

CpCV-E [AU:3487P:2002]

CpCV-E [AU:349Ma:2002]

CpCV-C [AU:3494Ib:2002]

CpCV-E [AU:3498K:2002]

CpCV-E [AU:3498A:2002]

CpCV-C [AU:3494Ia:2002]

TYDV-C [AU:3492Mb:2002]

CpCV-D [AU:2614a:2010]

TYDV-A [AU:2561:2010]TYDV-A [AU:2562:2010]

CpCV-E [AU:3495Mb:2002]

TYDV-A [AU:48:1985]

CpCV-D [AU:3494I:2002]

TYDV-A [AU:196:1986]

CpCV-B [AU:Qld21:2003]

CpYV [AU:3489B:2002]

CpCV-E [AU:3498Eb:2002]

CpCV-E [AU:84:1984]

CpCV-E [AU:3459G:2002]

CpCV-E [AU:3487H:2002]

CpCV-D [AU:2612:2010]

TYDV-A [AU:3492Ma:2002]

CpCV-A [AU:3494K:2002]

CpCV-E [AU:3458H:2002]

CpRLV [AU:Qld22:2003]

CpCV-D [AU:2614b:2010]

TYDV-A [AU:2564:2010]

CpCV-E [AU:3495Db:2002]

CpCV-D [AU:3768F:2003]

0.05

CpCDPKV [PK:ccdv3:2007]

BeYDV [PK:ccdv14:2005]

CpCDSV [SYR:SYR2:2008]

CpCDSDV [SD:Nhe5:1997]

BeYDV [SA]BeYDV [SA:mild]

CpCDSDV [SD:Eco18:1997]

CpCDPKV [PK:ccdv8:2007]CpCDPKV [PK:ccdv6:2005]

TYDV-A [AU:1992]

CpCV-A [AU:2683A:2002]

CpCV-E [AU:3460B:2002]

CpCV-A [AU:3459F:2002]

CpCV-E [AU:3489A:2002]

CpCV-E [AU:3498Ea:2002]

CpCV-E [AU:3495Da:2002]

CpCV-A [AU:3455C:2002]

CpCV-D [AU:2008:2007]

CpCV-E [AU:3487P:2002]

CpCV-E [AU:349Ma:2002]

CpCV-C [AU:3494Ib:2002]

CpCV-E [AU:3498K:2002]

CpCV-E [AU:3498A:2002]

CpCV-C [AU:3494Ia:2002]

TYDV-C [AU:3492Mb:2002]

CpCV-D [AU:2614a:2010]

TYDV-A [AU:2561:2010]

TYDV-A [AU:2562:2010]

CpCV-E [AU:3495Mb:2002]

TYDV-A [AU:48:1985]

TYDV-A [AU:196:1986]

CpCV-B [AU:Qld21:2003]

CpYV [AU:3489B:2002]

CpCV-E [AU:3498Eb:2002]CpCV-E [AU:84:1984]

CpCV-E [AU:3459G:2002]

CpCV-E [AU:3487H:2002]

CpCV-D [AU:2612:2010]

TYDV-A [AU:3492Ma:2002]

CpCV-A [AU:3494K:2002]

CpCV-E [AU:3458H:2002]

CpRLV [AU:Qld22:2003]

CpCV-D [AU:2614b:2010]

TYDV-A [AU:2564:2010]

CpCV-E [AU:3495Db:2002]

CpCV-D [AU:3768F:2003]

CpCV-D [AU:3494I:2002]

Euro-Asian streak viruses

African streak viruses

0.2SYR SYR

AFR

SYR

AFR

AFR

AUS

F cted am s detei

3g

iitamtvpt

aosseap

TNidvB

ig. 5. Maximum likelihood trees of the CP, Rep and MP proteins based on prediastreviruses which fell into divergent clades for CP and Rep, but not MP. Sequence

n this figure caption, the reader is referred to the web version of the article.)

.5. Selective forces acting on the dicot-infecting mastrevirusenes

Given that recombination patterns observable within the dicot-nfecting mastrevirus genomes mirror those seen in monocotnfecting mastreviruses such as PanSV and MSV we comparedhese three groups of viruses to determine whether their genes arelso evolving under similar selection pressures. Since the closestonocot-infecting mastrevirus relative of the dicot-infecting mas-

reviruses are the European dwarf viruses (EDVs) – Wheat dwarfirus, Barley dwarf virus and Oat dwarf virus – we also comparedatterns of natural selection detectable within this group of viruseso those detectable within the dicot-infecting mastreviruses.

Across all four virus groups it is apparent firstly that all genesre evolving under purifying selection (i.e. a lower proportionf non-synonymous substitutions are tolerable than synonymousubstitutions yielding dN/dS scores of less than 1; Table 2), and,

econdly, that the coat protein gene is evolving under the great-st degree of negative selection (i.e. mutations that result in aminocid substitutions are apparently generally less tolerable in the coatrotein than they are in other genes). Whereas in all of the analysed

able 2ormalised non-synonymous/synonymous substitution rate ratios within the cod-

ng regions of dicot-infecting mastreviruses compared with those of similarlyiverse groups of mastreviruses such as Maize streak virus (MSV), Panicum streakirus (PanSV), and the European dwarf viruses (EDV; including Wheat dwarf virus,arley dwarf virus and Oat dwarf virus).

Dataset Gene

Movement protein Coat protein Rep

Dicot 0.222441 0.186357 0.223076MSV 0.363271 0.137313 0.174095PanSV 0.270190 0.117245 0.142445EDV 0.247922 0.161192 0.188267

mino-acid alignments. African and Eurasian streak viruses are monocot-infectingrmined in this study are shown in red. (For interpretation of the references to color

monocot-infecting mastrevirus groups the gene evolving under thelowest degree of purifying selection is that encoding the movementprotein, it is apparent that dicot-infecting mastrevirus movementand replication associated proteins are both evolving under verysimilar intermediate degrees of purifying selection (Table 2).

It is important to point out that this small difference betweenthe synonymous and non-synonymous substitution rates observedbetween the monocot- and dicot-infecting mastreviruses almostcertainly does not reflect adaptation to dicot-infecting hosts (astage in the evolution of these viruses that obviously occurredbefore the most recent common ancestor of the dicot-infectingmastreviruses analysed here and which is therefore invisible to theanalyses that we have performed) but is rather reflective of contem-porary differences in the selective processes operating on monocot-and dicot-infecting mastreviruses. Given that dicot-infecting mas-trevirus movement protein genes have a lower non-synonymous:synonymous substitution rate ratio than any of the monocot-infecting mastreviruses whereas their Rep genes have a higher ratiothan that of these other viruses, it is plausible that the selectivelandscapes of both genes differ between monocot and dicot hosts.

3.6. Phylogenetic analysis of predicted amino acid sequences forRep, MP and CP proteins

The monocot-infecting mastreviruses assigned to differentspecies consistently clustered within monophyletic clades withinmaximum likelihood phylogenetic trees constructed using pre-dicted CP and Rep amino acid sequences (Fig. 5; pairwise distancecomparisons are provided in Supplementary Fig. 3). Possibly as

a result of some of the viruses being obviously recombinant, thetopologies of the Rep and CP trees differed quite substantiallyfrom that of the full genome tree (Fig. 2) with respect to branchesindicating the relationships between the different species. For

J. Hadfield et al. / Virus Resea

FeiQ

eatbrOvvothAeitmiMi

3

owwiotgaAniCw

4

stod

ig. 6. Phylogeographic distribution of the dicot-infecting mastrevirus isolates inastern Australia, with each symbol representing a viral strain or species. Multiplenfections of the same viral strain in a single plant are only represented once. Qld,ueensland; NSW, New South Wales; Vic, Victoria; WA, Western Australia.

xample, in the Rep tree, Australian TYDV and Afro-Asian CpCDVre sister taxa (recombination event 1, Fig. 2), whereas the Aus-ralian chickpea strains CpRLV, CpYV and CpCV formed a separateranch consistent with a common ancestral lineage based onecombination (events 8 and 9, respectively; Fig. 2). Althoughrosius orientalis has been identified as a vector for TYDV, noectors have been identified for the other dicot-infecting mastre-iruses from Australia. Since the CP is likely the sole determinantf geminiviral vector specificity (Briddon et al., 1990) and all ofhe Australian dicot-infecting mastreviruses other than CpYV haveighly conserved coat protein sequences it is likely that all theustralian viruses other than CpYV are also transmissible by O. ori-ntalis. Although in our MP tree it appears as though some dicotnfecting mastrevirus MP sequences (Fig. 5) are more closely relatedo the MP sequences of Eurasian and African monocot-infecting

astreviruses than they are to the MP sequences of other dicotnfecting mastreviruses it should be pointed out that the analysed

P sequences aligned very poorly between the groups and that its unclear where the root of the MP tree should be placed (Table 2).

.7. Phylogeographic distribution

Previous reports have indicated that the geographical rangesf distinct dicot-infecting mastreviruses are broadly overlappingithin eastern Australia (Schwinghamer et al., 2010). However,ith our larger sample we have found some evidence of geograph-

cal clustering of some species within Australia (Fig. 6). As has beenbserved previously for different MSV strains in sub-Saharan Africa,he CpCV-A, -B and -E strains all have broadly overlapping geo-raphical ranges across Eastern Australia with the range of CpCV-Elso extending to western Australia. Relative to CpCV in easternustralia, TYDV appears to have a more southerly, distribution thatevertheless overlaps substantially with that of CpCV-a fact that

s also evidenced by our observation that viruses belonging to thepCV-A -B and -C strains all bare traces of recombination eventsith TYDV.

. Concluding remarks

In this study we report from Eastern Australia one tentative new

pecies of a dicot-infecting mastrevirus (chickpea yellows virus),hree tentative new strains of CpCV and one tentative new strainf TYDV. We provide both a measure of dicot-infecting mastrevirusiversity in Eastern Australia and determine the degrees to which

rch 166 (2012) 13– 22 21

these viruses are exchanging genetic material through recombi-nation. With regard to the sequence diversity observed withinthe various species, the patterns of genetic exchange betweenthese species, patterns of natural selection and the generationduring infections of sub-genomic molecules, the dicot-infectingmastrevirus species bare a striking resemblance to their monocot-infecting counterparts from Africa and Eurasia.

Whereas monocot-infecting mastreviruses such as MSV havebeen studied extensively due to their socio-economic significancein sub-Saharan Africa (Varsani et al., 2008), dicot-infecting mas-treviruses have been studied considerably less primarily becausethey are considered a smaller present and future economic threat.Just as extensive sampling of MSV in cultivated and weed hosts(especially for the pathogenic MSV-A strain) has revealed its spreadthrough Africa (Monjane et al., 2011) and complex recombinant his-tory (Varsani et al., 2008), so too could a similar sampling regimeclarify the evolutionary origins of the Australian dicot-infectingmastreviruses.

Finally, although chickpea has only been commercially grownin eastern Australia since 1979, increased cultivation of chickpeasin the 1990s has meant that today in Australia there are 309,000 haunder this crop (Knights et al., 2007). Although substitution rateshave never been determined rigorously for dicot-infecting mas-treviruses, it is likely that they are similar to those observed formastreviruses like MSV and Sugarcane streak Reunion virus (i.e.∼2 × 10−4 substitutions per site per year; Harkins et al., 2009a,b).Unless the Australian dicot-infecting viruses are evolving ordersof magnitude faster than this rate, it seems highly unlikely thatthe current diversity of chickpea infecting Australian mastrevirusescould have evolved within the 30–40 years that chickpeas havebeen widely cultivated in Australia. This indicates firstly thatthese viruses naturally infect other hosts, as is known for TYDV(Thomas and Bowyer, 1984) and, secondly, that they are likelyonly beginning the process of adapting to infecting chickpea. Asurvey by Schwinghamer et al. (2010) revealed that there was ahigh incidence of dicot-infecting mastrevirus infection in plantsspecies such as subterranean clover, turnip weeds and guar. Asis likely to have happened in Africa where monocot-weed infect-ing mastreviruses adapted to infect and cause serious disease incultivated maize following its introduction there, indigenous Aus-tralian dicot-infecting mastrevirus species will probably adapt toinfect any newly introduced leguminous hosts. Therefore, whilstnot an immediate economic threat, the possibility remains thatserious disease causing dicot-infecting mastreviruses will in thefuture emerge to threaten Australian agriculture.

Acknowledgements

The molecular work was funded by the Marsden Fund of NewZealand (UOC0903) awarded to Arvind Varsani. Field collections in2002–2003 were funded by Industry & Investment NSW (formerlyNew South Wales Department of Primary Industries), The State ofQueensland Department of Employment, Economic Developmentand Innovation (DEEDI, formerly Queensland Department of Pri-mary Industries & Fisheries), and Grains Research and DevelopmentCorporation project DAN00023 awarded to Mark Schwinghamerand John E Thomas. Daisy Stainton was supported with a postgrad-uate scholarship from Marsden Fund of New Zealand (UOC0903).Simona Kraberger was supported by a School of Biological Sciences(University of Canterbury, New Zealand) postgraduate scholarship.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.virusres.2012.02.024.

2 Resea

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H

H

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