Genetic diversity and host range studies of turnip curly top virus

Sara Razavinejad • Jahangir Heydarnejad •

Mehdi Kamali • Hossain Massumi •

Simona Kraberger • Arvind Varsani

Received: 15 August 2012 / Accepted: 27 November 2012 / Published online: 9 December 2012

� Springer Science+Business Media New York 2012

Abstract Turnip curly top virus (TCTV) is a unique ge-

minivirus that has recently been characterised as infecting

turnips in Iran. The genome of TCTV shares \68 % pair-

wise identity with other geminiviruses and has a genome

organisation similar to that of curtoviruses and topocuvirus.

The replication-associated protein (Rep) bears the highest

similarity to curtovirus Reps (48.5–69.0 %); however, in

the case of the capsid protein (CP), the extent of similarity

is only 39.5–44.5 %. We constructed an agroinfectious

clone of TCTV and undertook host range studies on ten

plant species; in three species (turnip, sugar beet and

cowpea), we detected infection which presents curly top

symptoms in turnip and sugar beet. The efficiency of

TCTV infection in agroinoculated turnip plants was

71.7 %, and the infection was successfully transmitted to

80 % of the healthy turnip plants used in the insect trans-

mission studies by Circulifer haematoceps under green-

house conditions. We also determined the genome

sequence of 14 new TCTV isolates from southern Iran

isolated from turnips. We observed *13 % diversity

amongst all the TCTV isolates and found evidence of

recombination in the CP- and Rep-coding regions of the

genomes.

Keywords Turnip curly top virus � Curly top disease �Geminivirus � Iran

Introduction

The family Geminiviridae including geminate virions and

circular ssDNA genomes is potentially one of the largest

catalogued plant virus families. Based on the host range,

genome organisation and insect vector, geminiviruses are

classified into four genera namely Mastrevirus, Curtovirus,

Topocuvirus and Begomovirus [6]. Over the last five years,

a variety of novel viruses have been characterised, which

potentially represent new genera: Eragrostis curvula streak

virus (ECSV) [23], Beet curly top Iran virus (BCTIV) [2],

Turnip curly top virus (TCTV) [5], Grapevine cabernet

franc-associated virus (GCFaV) [14], and Citrus chlorotic

dwarf-associated virus (CCDaV) [16].

Briddon et al. [5] recently reported on TCTV, a novel

geminivirus from Iran, which has a genome consisting of

four overlapping genes in the complementary sense, similar

to those of topocurvirus and curtoviruses to some extent

(Fig. 1a). However, the virion sense encodes two genes

unlike curtoviruses which encode three, with capsid protein

(CP) sharing 39.5–44.5 % identity to those of curtoviruses.

Despite the homologous ORFs between TCTV and curt-

oviruses in the complementary sense, there is low sequence

identity except for C4 which shares *69.4 % amino acid

identity with curtoviruses. The C4 protein in curtoviruses is

a symptoms determinant protein, and it is not surprising

S. Razavinejad � J. Heydarnejad (&) � M. Kamali � H. Massumi

Department of Plant Protection, College of Agriculture, Shahid

Bahonar University of Kerman, Kerman, Iran

e-mail: jheydarnejad@mail.uk.ac.ir

S. Kraberger � A. Varsani

School of Biological Sciences, University of Canterbury, Private

Bag 4800, Christchurch 8140, New Zealand

A. Varsani

Biomolecular Interaction Centre, University of Canterbury,

Private Bag 4800, Christchurch 8140, New Zealand

A. Varsani

Electron Microscope Unit, University of Cape Town,

Rondebosch, Cape Town 7701, South Africa

123

Virus Genes (2013) 46:345–353

DOI 10.1007/s11262-012-0858-y

that the symptoms presented by TCTV infections are

indeed similar to those presented by curtoviruses. TCTV

induces curly top symptoms; in turnips, additional symp-

toms such as erection and curling inward of leaves, the

infected leaves becoming thick and brittle and swelling of

veins on the lower leaf surfaces with fluid being exuded

from the petioles or midribs on the lower leaf surfaces are

observed [5]. In addition to turnip, radish and four weed

species have been identified as natural hosts of TCTV, and

the virus has been shown to be transmitted by the leaf-

hopper, Circulifer haematoceps, under greenhouse condi-

tions [19]. In order to satisfy the Koch’s postulate, verify

that the symptoms observed in natural infection and

determine the host range of TCTV, we constructed an

infectious clone and undertook infection studies on ten

plant species. We also isolated 14 new TCTV isolates from

turnip samples from Fars province in southern Iran, which

we analysed for phylogenetic relationships, recombination

and basic strain classification.

Materials and methods

Construction of TCTV infectious clone

The TCTV-[IR:Zaf:B11:06] isolate (accession number

GU456685), sampled from turnip-growing farms in Zafar-

Abad (lat.: 29.4018, long.: 52.5838; 20 km south of Shiraz,

Fars Province, Iran) [5], showing inward leaf curling, thick

and brittle leaves, swelling of veins on the lower leaf

surfaces (Fig. 1b), was used as the virus source for con-

struction of an infectious clone.

TCTV cloned into pGEM�-3Zf(?) (Promega Biotech)

plasmid vector (2,981 nucleotides; designated as pGEM2.9)

was digested with KpnI and EcoRI restriction enzymes to

yield a 2,057-bp fragment of TCTV genome containing

replication origin. The 2,057-bp fragment was gel purified

by Qiaquick Gel Extraction Kit (Qiagen, Germany)

according to manufacturer’s protocol and cloned into the

binary vector pGreen0029 [11] restricted with KpnI and

EcoRI to generate a pGreen2.0 recombinant plasmid. A gel-

purified full-length TCTV genome obtained by digestion of

pGEM2.9 plasmid using KpnI restriction enzyme was

cloned into pGreen2.0 digested by the same enzyme. The

resulting plasmid, labelled pGreen5.038, containing partial

dimer of TCTV genome (full-length ? 2057 bp fragment;

Fig. 2a), was used to transform Escherichia coli cells (strain

JM107). Purified construct (pGreen5.038) and pSoup

(helper plasmid) were used simultaneously to transform

Agrobacterium tumefaciens strain C58 using the freeze/

thaw method according to Boulton [3].

Infectivity tests of TCTV construct

Transformed agrobacterium with pGreen5.038 and pSoup

plasmids were grown for 48–72 h at 28 �C on agar plates,

and the harvested bacterial cells were suspended in sterile

water. Inoculation of three leaves turnip (Brassica rapa L.

cv. White globe purple top), cabbage, sugar beet, spinach,

squash, cucumber, cowpea, datura, tobacco and tomato

seedlings (Table 1) were carried out by needle injection

with *10 ll of agrobacterium suspension at the crown,

near the surface of the soil, as described by Grimsley et al.

[10]. Second group of turnip seedlings were inoculated by

A. tumefaciens cultures bearing pGreen plasmid without a

TCTV partial dimer as negative control.

BCTIV

Rep (C1)

Rep (C1 + C2)

MP ? (V3)

Reg ? (V2)

CP (V1)

Rep (C1)

TCTV

Trap ? (C2)

CP (V1)

Ren (C3)

MP ? (V2)

(C4)?

Rep (C1)

(C4) ?

Trap ? (C2)

MP? (V2)

CP (V1)

Ren (C3)

Topocuvirus

Rep (C1)

SD (C4)reg(V2)

CP (V1)

Ren (C3)Trap/ss (C2)

Curtovirus

MP (V3)

A

Vector: Circulifer haematoceps Vector: Circulifer haematoceps

Vector: Circulifer tenellus Vector: Micrutalis malleifera

B

Fig. 1 a Genome organisation of curtoviruses, topocurvirus, BCTIV

and TCTV, b naturally infected turnip to Turnip curly top virus

showing leaf erection, inward rolling of leaf margin and swelling of

veins on the lower leaf surfaces

346 Virus Genes (2013) 46:345–353

123

Agroinoculated plants were kept in a greenhouse

(*12 h of light and *12 h of dark at 25 �C) and moni-

tored for curly top symptoms. Total DNA from inoculated

plants (either partial dimer construct or pGreen plasmid

without insert) was extracted 4 weeks post agroinoculation.

PCR assays were used to confirm TCTV infection in

agroinoculated plants coupled with visual curly top

symptoms presentation.

Total DNA from plant samples was extracted according

to the method described by Zhang et al. [24]. TCTV

infection of the samples was tested by PCR using two

primer pairs TCTV-840-F (GGCCAATGATGCTTTCTGG

KpnI(2430)

SacI(2563)

HindIII(185)

BglII(683)

NdeI(815)

EcoRI(1506)

KpnI(2430) HindIII(185)

BglII(683)

NdeI(815)

EcoRI(1506)

Full-length genome Partial-length genome

SacI(2563)

AFig. 2 a Schematic

representation of linear TCTV

partial dimer construct showing

potential stem loop structure

and cleavage sites of restriction

enzymes. Symptoms of

agroinoculated plants by TCTV

partial construct showing leaf

erection, inward leaf curling and

swelling of veins on the lower

leaf surfaces on turnip (b, c),

inward leaf curling and

production of swelling and

spine-like outgrowths of the

veins on the lower leaf surfaces

on sugar beet (d, e), pustule-like

(f), and necrotic spots (g) on

cowpea

Table 1 Characteristics of agroinoculated plant species by TCTV infectious construct, total number and number of TCTV infected plant

Common name Scientific name Family Number of infected

plant/number of

agroinoculated plants

Turnip Brassica rapa L. cv. White globe purple top Brassicaceae 43/60

Cabbage Brassica oleracea L. cv. capitata Brassicaceae 0/40

Sugar beet Beta vulgaris L. cv. Br1 Chenopodiaceae 2/30

Spinach Spinacia oleracea L. cv. Virofly Chenopodiaceae 0/30

Squash Cucurbita pepo L. cv. Maragheh Cucurbitaceae 0/40

Cucumber Cucumis sativus L. cv. Peto Seed Cucurbitaceae 0/30

Cowpea Vigna unguiculata L. cv. Mashhad Legominosae 15/35

Datura Datura maxima Sesse & Moc. Solanaceae 0/20

Tobacco Nicotiana Clevelendii Gray. Solanaceae 0/10

Tomato Lycopersicon esculentum Mill. cv. Queen Solanaceae 0/35

Virus Genes (2013) 46:345–353 347

123

GA)/TCTV-1594-R (AATATCATGGGACCACCAGGA)

and TCTV-97-F (CTTGGTCGTGTGGTCCCAAAGG)/

TCTV-1594-R which direct to amplify a 775-bp fragment

covering a part of V1, C2 and C3 ORFs and 1,517-bp

fragments covering complete V1, V2 and parts of C2 and

C3 ORFs of the TCTV genome. PCR conditions and

amplification conditions were used as described by

Heydarnejad et al. [12].

Leafhopper transmission of virus from agroinoculated

plants

Instar nymphs and/or adults of C. haematoceps collected

from sesame growing farms in Sirjan (180 km southwest of

Kerman province, Iran) were used for transmission study.

The method described by Heydarnejad et al. [12] was used

to obtain non-viruliferous leafhoppers from the established

colonies of these C. haematoceps. Different nymphal stages

and/or adults of non-viruliferous leafhoppers were used to

transmit TCTV from the infected agroinoculated turnip

plants to the healthy seedlings under greenhouse conditions

(*25 �C). The inoculated plants were examined for TCTV

infection 4 weeks later for curly top symptoms and by PCR

using two sets of primers described above.

Cloning and sequencing of new TCTV isolates

Fourteen turnip samples showing curly top symptoms were

collected from Zafar-Abad (n = 7), Homayejan (n = 4)

and Lapouei (n = 3) in the Fars province of southern Iran.

Total DNA was extracted from the leaves using CTAB

method as described by Zhang et al. [24]. The DNA was

then used as a template for rolling circle amplification

using TempliPhi Amplification Kit (GE Healthcare, USA)

as described by Shepherd et al. [20], and the resulting

concatemers were digested with BglII restriction endonu-

clease to yield unit length genomes. The unit length-

restricted fragments were cloned into a pUC19 plasmid

linearised with BamHI (BglII compatible cloning site). The

resulting clones were sequenced by primer walking at

Macrogen Inc. (South Korea).

Sequence analyses

The sequences of the TCTV isolates were assembled using

DNAMAN (v. 5.2.9). These were aligned with all available

sequences of curtoviruses, topocuvirus, other curto-like

sequences (BCTIV and Spinach curly top Arizona virus,

SCTAV) and ECSV (as an out-group for downstream

phylogenetic analysis) using MUSCLE [8]. Nucleotide

pairwise identities were calculated using SDTv 1.0 [18].

Full genome maximum likelihood phylogenetic trees were

constructed with the substitution model GTR?G4 (deter-

mined at the best-fit model by RDP 4 [17] using PHYML

3.0 [9] with 1,000 bootstrap replicates. Maximum likeli-

hood phylogenetic trees of the protein sequences were

constructed with PHYML 3.0 [9] using LG model of amino

acid substitutions with approximate likelihood-ratio test

(aLRT) for branch support [1]. Recombination analysis

was performed using RDP, BOOTSCAN, MAXCHI,

CHIMERA, SiScan and 3SEQ methods implemented in

RDP4 (v 4.13) [17].

Results

Agroinoculation of monopartite TCTV-cloned genome

Amongst agroinoculated plants, three plant species

including turnip, sugar beet and cowpea reacted to TCTV

inoculation of partial dimer construct, and symptoms

appeared. Typical TCTV symptoms including leaf erec-

tion, inward rolling of leaf margin and swelling of veins on

the lower leaf surfaces (Fig 2b, c) appeared in 71.7 %

(43/60) (Table 1) of the inoculated turnip plants. Symp-

toms were observed 10 days post agroinoculation on turnip

plants. The infection of symptomatic plants was confirmed

by PCR using TCTV-840-F/TCTV-1594-R and TCTV-97-

F/TCTV-1594-R primer pairs. While 775 and 1,517-bp

fragments were amplified in PCR using extracted DNA of

symptomatic plants, no symptoms were observed or TCTV

viral DNA detected in the negative control turnip plants,

inoculated with pGreen without insert.

Relatively lower infection rates of 6.7 % (2/30) were

observed in agroinoculated sugar beet plants with late

symptoms being observed at 6 weeks post agroinoculation.

These symptoms included inward leaf curling, swelling of

veins and vein enation on the lower leaf surfaces (Fig. 2d,

e). Agroinoculation of the cowpea plants resulted in

infection, 10 days post agroinoculation. Pustule-like and/or

necrotic spots were observed around the veins of cotyledon

leaves in 42.9 % (15/35) of the inoculated plants (Fig. 2f;

Table 1). The pustule-like spots gradually changed to

necrotic spots (Fig. 2g) but seldom extended to other

leaves.

Either TCTV viral DNA was not detected or symptoms

were not observed in the following: agroinoculated cab-

bage (Brassica oleracea L. cv. Capitata), spinach (Spinacia

oleracea L. cv. Virofly), squash (Cucurbita pepo L. cv.

Maragheh), cucumber (Cucumis sativus L. cv. Peto Seed),

Jimson weed (Datura maxima Sesse & Moc.), tobacco

(Nicotiana Clevelendii Gray.), and tomato (Lycopersicon

esculentum Mill. cv. Queen) plants.

348 Virus Genes (2013) 46:345–353

123

Vector transmission of TCTV-cloned genome

Circulifer haematoceps was demonstrated to transmit

TCTV (80 %; 16/20) from agroinoculated turnip plants at

to the healthy seedlings under greenhouse conditions

(*25 �C). Symptoms were observed 10 days after the start

of leafhopper feeding, and typical curly top symptoms

similar to those of agroinoculated plants were noted, and

the infection was confirmed by PCR.

Sequences analysis

The TCTV isolates are phylogenetically distinct from curt-

oviruses, topocuvirus, BCTIV and SCTAV. Genome-wide

pairwise identity analysis of the TCTV isolates (Fig. 3)

revealed that there was a diversity of 13 % (all isolates

share [87 % pairwise identity). Analysis of the distribution

of pairwise identities of TCTV (191 pairwise comparisons;

Fig. 3) revealed two regions of pairwise identity

representation: 87–94 %, and 99–100 %. Based on this, we

have established TCTV strain demarcation at 95 % pairwise

identity, and therefore, all TCTV isolates that share \95 %

pairwise identity belong to a new TCTV strain. Using the

above data, we have classified the 20 TCTV isolates into four

strains: TCTV-A, -B, -C, and -D (Table 2; Fig. 3).

A global pairwise identity analysis of TCTV, BCTIV/

SCTAV, topocuvirus and curtoviurses (Fig. 3) indicates

that the greatest diversity is observed within the curtovi-

rus genus (excluding TCTV, BCTIV and SCTAV) where

the members share [66.3 % pairwise identity. Interest-

ingly, TCTV isolates share 58.4–68.1, 57.3–61.6 and

61.5–62.6 % pairwise identities to curtoviruses, BCTIV/

SCTAV and topocuvirus, respectively. The isolates of the

two established genera, topocuvirus and curtoviruses,

share 58.7–65.3 % pairwise identity. Hence, based on our

phylogenetic analysis and pairwise identity comparisons,

and coupled with differences in genome organisation and

insect vectors, it is not surprising that Heydarnejad et al.

TCTV-A [KC108895]

TCTV-A [GU456689]

TCTV-A [GU456687]

TCTV-A [KC108894]

TCTV-A [GU456688]

TCTV-A [KC108893]

TCTV-A [GU456685]

TCTV-B [KC108896]

TCTV-B [KC108897]

TCTV-B [JQ742019]

TCTV-B [GU456686]

TCTV-B [KC108898]

TCTV-B [KC108899]

TCTV-B [KC108904]

TCTV-B [KC108900]

TCTV-C [KC108901]

TCTV-C [KC108906]

TCTV-C [KC108905]

TCTV-C [KC108903]

TCTV-D [KC108902]

88

90

92

94

96

98

100

KC

1088

95

GU

4566

89

GU

4566

87

KC

1088

9

GU

4566

88

KC

1088

93

GU

4566

85

KC

1088

96

KC

1088

97

JQ

7420

19

GU

4566

86

KC

1088

98

KC

1088

99

KC

1089

04

KC

1089

00

KC

1089

04

KC

1089

06

KC

1089

05

KC

1089

03

KC

1089

02

>90%

60–89%

TCTV

Curtoviruses

Bootstrap support

0.05 substitutions per site

BCTIV / SCTAV (Becurtoviruses)

Topocuvirus

87 88 89 90 91 92 93 94 95 96 97 98 99 1000.00

0.05

0.10

0.20

0.30

0.35

0.15

0.25

Strains Variants

Percentage pairwise identities

Pro

po

rtio

n o

f p

airw

ise

iden

titi

e s

ECSV [FJ665629]

TPCTV [X84735] SpCTV [AY548948]

PeCTV [EF501977] BSCTV [X97203]

BSCTV [U02311] BSCTAV [FJ545686]

BMCTV [U56975] BMCTV [AY134867]

BMCTV [HQ634913] BMCTV [EU586260] BMCTV [HQ214016] BMCTV [EU586261] BMCTV [EU193175]

BCTV [M24597] BCTV [AF379637]

SpSCTV [GU734126] HrCTV [U49907]

SCTAV [HQ443515]

BCTIV0.1 substitutions per site

Per

cent

age

pairw

ise

nucl

eotid

e id

entit

y

A

D

PeYDV [EU921828]

TCTV

BCTIV / SCTAVTopocuvirus

Curtoviruses

TCTV 86.8 - 99.9 %

BCTIV / SCTAV 57.3 - 61.6 % 76.6 - 99.9 %

Topocuvirus 61.5 - 62.6 % 55.4 - 58.7 % 100 %

Curtoviruses 58.4 - 68.1 % 63.8 - 68.7% 58.7 - 65.3 % 66.3 - 98.7%

C

B

Fig. 3 a Maximum likelihood phylogenetic tree showing the evolu-

tionary relationship of TCTV with curtoviruses, topocuvirus, BCTIV

and SCTAV, with ECSV being used to root the tree, b enlargement of

maximum likelihood phylogenetic relationship of the TCTV isolates

with a two dimensional percentage pairwise identity plot. TCTV

isolates determined in this study are in bold, c percentage pairwise

identity comparisons of genomes of TCTV, BCTIV/SCTAV, topoc-

uvirus and curtovirus isolates, d distribution of pairwise identities of

TCTV isolates

Virus Genes (2013) 46:345–353 349

123

[12] and Briddon et al. [5] proposed that BCTIV and

TCTV be considered as members of new genera (Fig. 3a,

b, c, d).

We found evidence for two putative recombination

events amongst the TCTV isolates. TCTV-B and –D are

recombinants with a domain in the CP region which is most

similar to that of TCTV-C (Fig. 4), whereas TCTV-B is a

double recombinant with a domain spanning the Rep that is

most similar to that of TCTV-C and -D (Fig. 4). Maximum

likelihood phylogenetic analysis of the CP and Rep further

supports this evidence (Fig. 4). TCTV CPs share \44.6,

44.6 and 22.9 % amino acid identity to the CPs of curtovi-

ruses, BCTIV/SCTAV and topocuvirus, respectively

(Fig. 5). On the other hand, the TCTV Reps share \68.9,

30.9 and 62.1 % amino acid identity with the Reps of curt-

oviruses, BCTIV/SCTAV and topocuvirus, respectively.

Discussion

In Iran, three geminiviruses associated with curly top dis-

eases: BSCTV [4], BCTIV [2] and TCTV [5] have been

reported in sugar beet or turnip-growing farms. The bio-

logical properties of TCTV are most similar to those of

members of the genus Curtovirus; however, the genome

organisation is most similar to that of topocuvirus. Further,

phylogenetically, TCTV is distinct and monophyletic [5].

The typical curly top symptoms, leafhopper transmission

by C. haematoceps aligns these characteristics with those

of BCTIV [2, 5, 13, 19]; however, in contrast to BCTIV,

we found TCTV had almost no infectivity in sugar beet

(6.7 % efficiency). These results corroborate with obser-

vations made in surveys of TCTV infection in turnip farms

in Homayejan (50 km west of Shiraz, Fars province, Iran)

where sugar beet was infected with BCTIV [19], thereby

suggesting that sugar beet plants are not a TCTV suitable

host. Our results reveal the limited host range of TCTV

which is similar to that of Horseradish curly top curtovirus

(HrCTV) [15], which, similar to, Spinach severe curly top

virus (SpSCTV) shares only 67.1–70.6 % pairwise identity

with other curtoviruses. Amongst ten plant species agro-

inoculated with TCTV construct, only three plant species

(Table 1) were infected. Furthermore, attempts to find

possible additional hosts of the virus within or around the

severely TCTV-infected farms led to only the virus being

detected in symptomatic radish plants (genome accession #

JQ742019) [19] and four symptomless weed species.

Despite numerous attempts, we were unable to recover any

full TCTV genomes from the four symptomless weed

samples. Nevertheless, more studies are necessary to

identify natural hosts of the virus in cultivated crops and

possible reservoirs in uncultivated plants.

The genome of curtoviruses comprises 6–7 ORFs, three

encoded in the virion sense and 3–4 in the complementary

sense, which separated by one IR [6] and curtoviruses, are

transmitted by the leafhopper Circulifer tenellus (Baker)

Table 2 Details of TCTV isolates, hosts and accession numbers. Isolates determined in this study are in bold

GenBank accession Field id Sampling year Host Location

GU456685 TCTV-A [IR:Zaf:B11:06] 2006 Brassica rapa Zafar Abad, Fars province (southern Iran)

GU456686 TCTV-B [IR:Hom1:2K:09] 2009 Brassica rapa Homayejan, Fars province (southern Iran)

GU456687 TCTV-A [IR: Hom2:8K:09]: 2009 Brassica rapa Homayejan, Fars province (southern Iran)

GU456688 TCTV-A [IR: Hom3:4K:09] 2009 Brassica rapa Homayejan, Fars province (southern Iran)

GU456689 TCTV-A [IR: Hom3:7K:09] 2009 Brassica rapa Homayejan, Fars province (southern Iran)

JQ742019 TCTV-B [IR:Hom:T57K:Tur:10] 2010 Raphanus sativus Homayejan, Fars province (southern Iran)

KC108893 TCTV-A [IR:Lap:L13:Tur:12] 2012 Brassica rapa Lapoui, Fars province (southern Iran)

KC108894 TCTV-A [IR:Zaf:Z8:Tur:12] 2012 Brassica rapa Zafar Abad, Fars province (southern Iran)

KC108895 TCTV-A [IR:Lap:L2-P:Tur:12] 2012 Brassica rapa Lapoui, Fars province (southern Iran)

KC108896 TCTV-B [IR:Lap:L16:Tur:12] 2012 Brassica rapa Lapoui, Fars province (southern Iran)

KC108899 TCTV-B [IR:Hom:H6:Tur:12] 2012 Brassica rapa Homayejan, Fars province (southern Iran)

KC108898 TCTV-B [IR:Zaf:Z2-14:Tur:12] 2012 Brassica rapa Zafar Abad, Fars province (southern Iran)

KC108900 TCTV-B [IR:Zaf:Z2-18:Tur:12] 2012 Brassica rapa Zafar Abad, Fars province (southern Iran)

KC108897 TCTV-B [IR:Zaf:Z9:Tur:12] 2012 Brassica rapa Zafar Abad, Fars province (southern Iran)

KC108904 TCTV-B [IR:Hom:H8:Tur:12] 2012 Brassica rapa Homayejan, Fars province (southern Iran)

KC108905 TCTV-C [IR:Zaf:Z2-1:Tur:12] 2012 Brassica rapa Zafar Abad, Fars province (southern Iran)

KC108906 TCTV-C [IR:Zaf:Z5-2:Tur:12] 2012 Brassica rapa Zafar Abad, Fars province (southern Iran)

KC108901 TCTV-C [IR:Hom:H1:Tur:12] 2012 Brassica rapa Homayejan, Fars province (southern Iran)

KC108903 TCTV-C [IR:Zaf:Z10:Tur:12] 2012 Brassica rapa Zafar Abad, Fars province (southern Iran)

KC108902 TCTV-D [IR:Hom:H9:Tur:12] 2012 Brassica rapa Homayejan, Fars province (southern Iran)

350 Virus Genes (2013) 46:345–353

123

[7, 21, 22]. The TCTV genome has six ORFs, two in viral and

four in complementary strand. Despite having different

genome arrangements in the complementary sense ORFs,

products of all TCTV ORFs, other than V2, are most similar

to the counterpart ORFs of curtoviruses. However, the

complementary-sense ORFs of TCTV other than C4 show

little sequence similarity to their curtovirus homologs. Our

analysis of TCTV isolates revealed that there is *13 %

diversity, and, as with all other geminiviruses, recombina-

tion seems to play a key role in its evolution. The CP of all the

curtoviruses share [80.2 % amino acid identity; however,

they only bear an amino acid identity of 39.5–44.5 % to

those of TCTV. Nevertheless, the Reps of curtoviruses

share [53.1 % amino acid identity; whereas they share

48.5–69.0 % amino acid identity to TCTV Reps, clearly

suggesting that TCTV has a highly divergent CP. It is

interesting to note that in general there is high diversity in the

Rep of curtoviruses relative to TCTV; we presume this to be

as a result of recombination. It is highly likely, that when

coupled with new molecular tools and next generation

sequencing technologies, novel curto-like viruses will be

discovered from the Middle East which seems to be the

centre of curto-like virus diversity in contrast to the New

World, where most of the curtoviruses have been found.

Broad turnip survey in the Middle East will give us a better

indication of the incidence and diversity of TCTV.

PeYDV [EU921828]

BCTIV [EU273817]

BCTIV [EU273817]

PeYDV [EU921828]

0.5 amino acid substitutions per siter

>90%

60–89%

TCTV

Curtoviruses

Bootstrap support

BCTIV / SCTAV (Becurtoviruses)

Topocuvirus

ECSV [FJ665629] TPCTV [X84735]

SpCTV [AY548948]

PeCTV [EF501977]

BSCTV [X97203] BSCTV [U02311]

BSCTAV [FJ545686]

BMCTV [U56975] BMCTV [AY134867] BMCTV [HQ634913] BMCTV [EU586260] BMCTV [HQ214016]

BMCTV [EU586261] BMCTV [EU193175]

BCTV [M24597] BCTV [AF379637]

SpSCTV [GU734126]

HrCTV [U49907] SCTAV [HQ443515]

ECSV [FJ665629] TPCTV [X84735]

SpCTV [AY548948]

PeCTV [EF501977]

BSCTV [X97203] BSCTV [U02311]

BSCTAV [FJ545686]

BMCTV [U56975] BMCTV [AY134867]

BMCTV [HQ634913] BMCTV [EU586260]

BMCTV [HQ214016]

BMCTV [EU586261] BMCTV [EU193175]

BCTV [M24597] BCTV [AF379637]

SpSCTV [GU734126]

HrCTV [U49907]

SCTAV [HQ443515]

TCTV-A [KC108895] TCTV-A [GU456689] TCTV-A [GU456687]

TCTV-A [KC108894 ]

TCTV-A [GU456688]TCTV-A [KC108893] TCTV-A [GU456685]

TCTV-B [KC108896]

TCTV-B [KC108897 ]

TCTV-B [JQ742019]TCTV-B [GU456686]

TCTV-B [KC108898 ]

TCTV-B [KC108899 ] TCTV-B [KC108904 ]

TCTV-B [KC108900 ]

TCTV-C [KC108901] TCTV-C [KC108906] TCTV-C [KC108905 ]

TCTV-C [KC108903]

TCTV-D [KC108902 ]

TCTV-A [KC108895]

TCTV-A [GU456689]

TCTV-A [GU456687] TCTV-A [KC108894 ]

TCTV-A [GU456688]

TCTV-A [KC108893]

TCTV-A [GU456685]

TCTV-B [KC108896] TCTV-B [KC108899 ]

TCTV-B [JQ742019]

TCTV-B [GU456686]

TCTV-B [KC108898 ] TCTV-B [KC108899 ]

TCTV-B [KC108904 ]

TCTV-B [KC108900 ]

TCTV-C [KC108901] TCTV-C [KC108906] TCTV-C [KC108905 ]TCTV-C [KC108903]

TCTV-D [KC108902 ]

0.5 amino acid substitutions per siter

RepCP

BC

TCTV-BTCTV-D

Recombinant

TCTV-C

Potential major parents

Potential minor parent

TCTV-A

Detection methods

RGBMCT

P-value

1.8 x 10- 16

TCTV-B TCTV-CTCTV-D

TCTV-A RGBMCT 2.8 x 10 - 28

Event

II I

I

II

V2 (Mp)

Cp C4

Rep

C2 (trap)

Ren

A

Fig. 4 a Iillustration and details of the recombination events detected

by RDP 4. R, G, B, M, C, S and T indicate detection by the RDP

GENCONV, BOOTSCAN, MAXCHI, CHIMERA, SISCAN and

3SEQ methods, respectively (p value determined by the method

indicated in bold type), b maximum likelihood phylogenetic analysis

of the CP and Rep amino acid sequences of TCTV, curtoviruses,

topocurvirus, BCTIV, SCTAV (ECSV was used to root the CP

phylogenetic tree and BCTIV and SCTAV were used to root the Rep

phylogenetic tree). TCTV isolates determined in this study are in bold

Virus Genes (2013) 46:345–353 351

123

GenBank Accession #

KC108893: TCTV-A [IR: Lap13:Tur:12]

KC108894: TCTV-A [IR: Zaf8:Tur:12]

KC108895: TCTV-A [IR: Lap2-P:Tur:12]

KC108896: TCTV-B [IR: Lap16:Tur:12]

KC108897: TCTV-B [IR: Zaf9:Tur:12]

KC108898: TCTV-B [IR: Zaf2-14:Tur:12]

KC108899: TCTV-B [IR: Hom6:Tur:12]

KC108900: TCTV-B [IR: Zaf2-18:Tur:12]

KC108901: TCTV-C [IR: Hom1:Tur:12]

KC108902: TCTV-D [IR: Hom9:Tur:12]

KC108903: TCTV-C [IR: Zaf10:Tur:12]

KC108904: TCTV-B [IR: Hom8:Tur:12]

KC108905: TCTV-C [IR: Zaf2-1:Tur:12]

KC108906: TCTV-C [IR: Zaf5-2:Tur:12]

Acknowledgments This study was supported by a grant from

Shahid Bahonar University of Kerman, Kerman, Iran and a block

grant from the University of Cape Town, South Africa, awarded to

Arvind Varsani.

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ge a

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tovi

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s

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