Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India

G Model

ARTICLE IN PRESS

VIRUS 96547 1–9

Virus Research xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Virus Research journal homepage: www.elsevier.com/locate/virusres

Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India

1

2

Q1

3

Ashish Srivastava, Susheel Kumar, Meraj Jaidi, S.K. Raj ∗ Plant Molecular Virology, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, Uttar Pradesh, India

4 5

a r t i c l e

6 21

i n f o

a b s t r a c t

7

Article history: Received 31 December 2014 Received in revised form 10 February 2015 Accepted 14 February 2015 Available online xxx

8 9 10 11 12 13

20

Keywords: Jatropha species Yellow mosaic disease New begomovirus species DNA-A genome Sequence identity Phylogenetic relationships

22

1. Introduction

14 15 16 17 18 19

Severe yellow mosaic disease was observed in three ornamental species of Jatropha: J. integerrima, J. podagrica and J. multifida grown in gardens at Lucknow, India, during a survey in 2013. The causal pathogen was successfully transmitted from diseased to healthy plants of these species by whitefly (Bemisia tabaci). The infection of begomovirus was initially detected in naturally infected plant samples by PCR using begomovirus universal primers. The begomovirus was characterized having a monopartite genome based on sequence analyses of the cloned ∼2.9 kb DNA-A genome amplified by rolling circle amplification using Phi-29 DNA polymerase. The genome contained 2844 nucleotides that could be translated into seven potential open reading frames. The nucleotide sequences of DNA-A genome of the begomovirus isolates: JI (KC513823), JP (KF652078) and JM (KF652077) shared 94–95% identities together and 93–95% identities with an uncharacterized begomovirus isolated from J. curcas (the only sequences available in GenBank database as GU451249 and EU798996 under the name Jatropha leaf curl virus). These shared highest identity of 61% and highly distant phylogenetic relationships with other begomoviruses reported worldwide. Based on 61% sequence identities (much less than 89%, the species demarcation criteria for a new begomovirus) the isolates under study were identified as members of a new begomovirus species for which the name was proposed as “Jatropha mosaic Lucknow virus (JMLV)”. The recombination analysis also suggested that JMLV was not a recombinant species, hence considered as unidentified begomovirus species. Koch’s postulates were also established by agroinfiltration assay of agroinfectious clone of JMLV. Characterization of JMLV associated with yellow mosaic disease of J. integerrima, J. podagrica and J. multifida is being reported for the first time. © 2015 Published by Elsevier B.V.

Q3

Jatropha, a genus of perennial flowering plants of the family Euphorbiaceae, is native to Central America and distributed in Africa and Asia (Speight and Singh, 2014). The genus Jatropha includes various species such as Jatropha curcas, J. gossypifolia, J. integerrima, J. podagrica and J. multifida. Amongst them, J. curcas is widely cultivated as a major source of bio-fuel (Openshaw, 2000; Pramanik, 2003) while J. gossypifolia grows as a weed along road sides (Snehi et al., 2011a). The species J. integerrima, J. podagrica and J. multifida are grown as ornamental plants in Indian gardens. J. integerrima is an ornamental shrub that grows commonly in south parts of India (Sharma and Singh, 2013). J. podagrica is an ornamental plant is commonly grown in Australia, the Hawaiian Islands, Southern Africa, Mozambique, Zambia and warmer parts of Asia (Hooker

23 24 25 26 27 28 29 30 31 32 33 34 35

Q2

∗ Corresponding author. Tel.: +91 522 2297950; fax: +91 0522 2205836. E-mail address: [email protected] (S.K. Raj).

et al., 1848; Ojewole and Odebiyi, 1980). J. multifida is a very attrac- Q4 tive and widely cultivated species throughout the tropics, in north Australia and South east Africa (Dehgan, 1982; Nayak and Patel, 2009). Besides this, J. integerrima is traditionally used as purgative, styptic, emetic, in treatment of warts, tumors, rheumatism, herpes, pruritis, toothaches, scabies, eczema and ringworm (Kirtikar and Basu, 2002). Begomoviruses of the family Geminiviridae are whitefly transmitted and cause diseases of important crops in the tropics and subtropics (Tiwari et al., 2013). Their genome consists of one or two circular single stranded DNA components, referred to as DNA-A and DNA-B, each approximately of 2.6–2.8 kb in size (King et al., 2011; Fauquet et al., 2008). DNA-A encodes replication-associated protein (AC1) essential for viral replication; replication enhancer protein (AC3); transcriptional activator protein (AC2) that transactivates expression of AV1 and BV1 ORFs; AC4 protein; coat protein (AV1) for encapsidation and insect transmission, and precoat protein (AV2) for virus accumulation and symptom development, while AV2 ORF is missing in New World begomoviruses. The functions

http://dx.doi.org/10.1016/j.virusres.2015.02.015 0168-1702/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

G Model VIRUS 96547 1–9

ARTICLE IN PRESS A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

2

86

of AC4 protein are not proven for all the begomoviruses. However, some functions of C4 protein were shown only in case of monopartite begomoviruses (Hanley-Bowdoin et al., 2013). The DNA-B encodes the nuclear shuttle protein (NSP; BV1) and movement protein (MP; BC1), both of which determine viral pathogenicity, but the coat protein in case of a defective NSP can rescue the function of NSP (Hanley-Bowdoin et al., 2013; Jeske, 2009). A number of begomoviruses occurring in the Old World (Eastern Hemisphere, Europe, Africa, Asia) are monopartite and have only a single component equivalent to DNA-A. The cloned genomic component of some of these monopartite begomoviruses have been shown to produce typical symptoms, confirming that single genomic component was solely responsible for the disease (Zhang et al., 2010; Kon and Gilbertson, 2012). A satellite molecule (betasatellite) has also been shown to be associated with mono- and bi-partite begomoviruses, and may be required for systemic infection and symptom development (Saunders et al., 2000; Briddon et al., 2003; Srivastava et al., 2013, 2014a; Simmonds-Gordon et al., 2014). The occurrence of begomoviruses has been noted on J. curcas and J. gossypifolia species worldwide (Aswatha-Narayana et al., 2006, 2007; Tewari et al., 2007; Raj et al., 2008; Gao et al., 2010; Ramkat et al., 2011a,b; Snehi et al., 2011a,b, 2012; Appiah et al., 2012; Kashina et al., 2013; Srivastava et al., 2014b). African cassava mosaic virus (syn. Cassava latent virus) from Kenya (Bock et al., 1981) and Jatropha mosaic virus from Florida (Polston et al., 2014) have also been reported on J. multifida. However, no virus on J. integerrima and J. podagrica has been reported hitherto. In this communication, the association of an unidentified begomovirus with yellow mosaic disease of J. integerrima, J. podagrica and J. multifida has been investigated for the first time based on the sequence analyses of complete DNA-A genome.

87

2. Materials and methods

88

2.1. Surveys and collection of plant material

55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

et al., 2005). PCR was set up in a 50 ␮l reaction mixture containing: template DNA (100 ng), dNTPs (200 ␮M each), primers (each 0.25 ␮M), MgCl2 (1.5 mM), Taq DNA polymerase (1.0 U, Bangalore Genei Pvt. Ltd.), Taq buffer (1X, Genei Pvt. Ltd., Bangalore, India) in a Peltier thermal cycler PTC200 engine (MJ Research, Waltham, MA, USA). PCR conditions included initial denaturation at 94 ◦ C for 5 min; followed by 30 cycles of denaturation at 94 ◦ C for 1 min; annealing at 52 ◦ C for 1 min and extension at 72 ◦ C for 1 min 30 s and a final extension at 72 ◦ C for 5 min. The PCR was also performed using DNA-B specific primers (BC1F and BC1R; Padidam et al., 1995) to detect DNA-B genome. The PCR conditions were same as described for DNA-A amplification. For detection of betasatellite association with begomovirus, PCRs were carried out with betasatellite specific primers (␤-01 and ␤-02; Briddon et al., 2003) using the total DNA from same samples. The PCR conditions were again same as described earlier. All PCR products were analyzed by electrophoresis on 1.0% agarose gel using a 1 kb DNA ladder (Thermo Fisher Scientific Inc., Pittsburgh, United States) as size markers. 2.4. Rolling circle amplification, cloning and sequencing of complete begomoviral genome For molecular characterization of the detected begomovirus, the complete begomoviral genomes were amplified from the DNA isolated from infected J. integerrima, J. podagrica and J. multifida samples by rolling circle amplification (RCA) using Phi-29 DNA polymerase enzyme as per the manufacturer’s instructions (TempliPhiTM DNA amplification kit, GE Healthcare, USA). The reaction was stopped by incubating the mixture at 65 ◦ C for 10 min and the RCA products were monomerized by digestion with BamHI restriction enzymes (New England BioLabs, USA) and the digested products were electrophoresed on 1% agarose gel. The expected amplicons obtained were purified using Wizard SV Gel & PCR CleanUp System (Promega Corporation, Madison, USA) and ligated into pCAMBIA1300 vector at BamHI site. Competent cells of DH5␣ strain of Escherichia coli were transformed using the ligated mixture by the heat shock method (Sambrook and Russel, 1989). Three positive clones of each Jatropha species were sequenced by primer walking (Genei Pvt. Ltd., Bangalore, India). Consensus sequence data were deposited in the GenBank database.

93

The surveys were conducted in January and May of 2013 on J. integerrima, J. podagrica and J. multifida species growing in the gardens at Lucknow (Lat. 26◦ 55 N Lon. 80◦ 59 E), India. The leaf samples from severely diseased plants (three samples from each species) were collected for experiments.

94

2.2. Virus transmission through whitefly

2.5. Analysis of sequence data of begomoviral genome

Whitefly transmission tests were performed using the nonviruliferous B. tabaci maintained on healthy Nicotiana tabaccum cv. White Burley plants. The adult B. tabaci were collected, starved for 2 h and allowed to feed separately on naturally infected J. integerrima, J. podagrica and J. multifida plants showing yellow mosaic symptoms for an acquisition access period of 24 h. The viruliferous B. tabaci were transferred onto healthy seedlings (10–12 per plant) of J. integerrima, J. podagrica, J. multifida, Solanum lycopersicum and N. glutinosa, and were allowed an inoculation access period of 24 h. Then the whiteflies were killed by spraying with insecticide (Confidor, Bayer CropScience Ltd., Mumbai, India) and the inoculated plants were maintained in an insect proof glasshouse for two months to monitor for symptoms.

The sequence data from the begomovirus isolates were analyzed by BLASTN (http://www.ncbi.nlm.nih.gov/BLAST) and compared with existing sequences of begomovirus strains available in the GenBank database. The open reading frames (ORFs) were checked by ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and translated into amino acids using the Expasy tool (http://www.expasy.org/tools/dna.html). The matrix for pairwise alignment of selected begomovirus isolates was obtained using Clustal-W method of the Genomatix DiAlign 2 program (Morgenstern et al., 1998). The 45 amino acids at the C-terminus of the coat protein of begomovirus were also aligned using Genomatix DiAlign 2 program (Morgenstern et al., 1998). Phylogenetic analyses were performed using the Molecular Evolutionary Genetics Analysis tool, MEGA v6.0 (Tamura et al., 2013) with 1000 replicates bootstrapping, and the dendrograms were generated with the Neighbour joining (NJ) method and viewed by the NJ plot program. The phylogenetic trees of coat proteins (CP) and replication associated proteins (Rep) of begomovirus and other genera of the family Geminiviridae obtained from BLASTP database were also constructed using Maximum Likelihood method conducted in MEGA v6.0 (Tamura et al., 2013).

89 90 91 92

95 96 97 98 99 100 101 102 103 104 105 106 107

108

109 110 111 112 113

2.3. Molecular detection of begomovirus in Jatropha species For initial detection of the begomovirus, the total DNAs were isolated from newly emerging leaves of infected plants (J. integerrima, J. podagrica and J. multifida) by the method of Dellaporta et al. (1983). PCRs were performed using a pair of begomovirus DNA-A specific universal primers: PALIv 722/PALIc 1960 (Reddy

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

133 134

135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152

153

154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174

G Model VIRUS 96547 1–9

ARTICLE IN PRESS A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

175

2.6. Agroinoculation assay

To determine the infectivity of begomovirus DNA genome, 177 the infectious clones were generated into pCAMBIA1300 vector 178 backbone. For infectious clone preparation, the genome of JM 179 (J. multifida) isolate was digested with SalI and BamHI enzymes 180 and 1624 bp fragment was ligated in pCAMBIA1300 vector at 181 SalI/BamHI site and E. coli (DH5␣ strain) cells were transformed. 182 The positive clones were again digested with BamHI enzyme and 183 ligated into BamHI digested complete 2844 nt fragment in such 184 a way that the IR regions should be in the same orientation. The 185 orientation of the infectious clones was determined by sequence 186 analysis of their sequence data. 187 The verified clones were mobilized into Agrobacterium tume188 faciens strain GV3101 containing pMP90 helper plasmid and was 189 designated as pCAM-JM-A while pCAMBIA1300 (pCAM) alone was 190 also transformed as a negative control. The cultures were sus191 pended in MES buffer to 1.0 OD and infiltrated into lower surface 192 of leaves of J. integerrima, J. podagrica, J. multifida, S. lycopersicum, 193 N. glutinosa and N. benthamiana plants in three independent sets 194 (three plants in each set) along with a negative control (pCAM) 195 using 2 ml syringe by pressure infiltration method (Jia and Fang, 196 Q5 2003) in triplicate. Agroinfiltrated plants were maintained in insect 197 proof glasshouse and monitored for two months for symptom 198 development. For confirmation of viral genomes in distant leaves, 199 total DNA was isolated and subjected to PCR using begomovirus 200 specific primers (Reddy et al., 2005) and the PCR products were 201 analyzed by electrophoresis on 1.0% agarose gel. 176

202

2.7. Recombination analysis of the begomoviral genome

To perform recombination analysis, the nucleotide sequence 204 data of begomovirus isolates along with other begomoviruses 205 of the family Geminiviridae were aligned by Clustal-W and 206 analyzed using the RDP, GENECONV, BOOTSCAN, MAXCHI, 207 CHIMAERA, SISCAN and 3SEQ methods implemented in RDP 208 Q6 v4.33 (Martin et al., 2010). In addition, the begomoviral 209 genomes were analyzed using the Simplot program v3.2 210 (http://sray.med.som.jhmi.edu/SCRoftware/Simplot), with a slid211 ing window of 200 nucleotides moving in 20-nucleotide steps. 203

212

3. Results

213

3.1. Disease symptoms and whitefly transmission tests

226

During the surveys in January and May 2013, a yellow mosaic disease was observed on J. integerrima, J. podagrica and J. multifida plants growing in gardens at Lucknow, India. The infected plants showed severe to mild yellow mosaic symptoms on leaves and stunting of plants, compared to the healthy controls (Fig. 1). During whitefly transmission, the causal virus from each of the three Jatropha species was successfully transmitted from diseased to healthy seedling of J. integerrima, J. podagrica and J. multifida that induced yellow mosaic symptoms by 35 dpi (Table 1) similar to naturally infected plants. The inoculated N. glutinosa and S. lycopersicum plants also developed mild yellow mosaic symptoms by 35 dpi (Table 1) indicating positive transmission of the begomovirus in these test species.

227

3.2. Molecular detection of begomovirus

214 215 216 217 218 219 220 221 222 223 224 225

228 229 230 231 232

PCR amplification using PALIv 722/PALIc 1960 primers and total DNA isolated from infected J. integerrima, J. podagrica and J. multifida samples resulted in the expected size amplicon of ∼1.2 kb from all these samples, similar to a positive control (experimentally inoculated J. curcas by a well characterized begomovirus, Snehi

3

et al., 2012). No such band was observed from the healthy plant samples. These results indicated presence of the begomovirus in symptomatic J. integerrima, J. podagrica and J. multifida samples. Also, no amplicons were obtained in PCRs performed either with DNA-B genome specific or betasatellite specific primers as against when the cloned partial DNA-B (tomato leaf curl New Delhi virus; DQ873412) and betasatellite (ageratum leaf curl betasatellite; JQ408218) were taken as positive control for DNA-B and betasatellite, respectively. The negative PCR suggested that the begomovirus associated with the yellow mosaic disease may be a monopartite begomovirus and may not have any associated satellite molecule. The whitefly inoculated plants were also tested by PCR using the same primers and gave positive amplification of the ∼1.2 kb band in all inoculated samples of J. integerrima, J. podagrica and J. multifida as well as N. glutinosa and S. lycopersicum (Table 1), suggesting successful transmission of the begomovirus to these plant species. 3.3. Genome organization of the begomovirus The electrophoresis of RCA products obtained from DNA of three infected J. integerrima, J. podagrica and J. multifida samples (one representative of each species) followed by their restriction digestion with BamHI resulted in the single amplicon of ∼2.9 kb in all samples. The amplicons obtained were gel purified and ligated separately in pCAMBIA1300 vector at BamHI site and three positive clones of each species were sequenced. The sequence data of these clones were found 100% identical, therefore, only one sequence of each Jatropha species was submitted in GenBank database as KC513823 of J. integerrima (JI), KF652078 of J. podagrica (JP) and KF652077 of J. multifida (JM) isolate. Sequence analysis revealed presence of the full length genome of 2844 nucleotides for the JI, JP and JM isolates, which contained seven potential ORFs: V1 and V2 in virion sense, and C1, C2, C3, C4 and C5 in complementary sense. The V2 and V1 ORFs were located at the position from 175-528 nt (117 aa) to 335-1072 nt (245 aa) respectively, and encoding the pre-coat protein and coat protein, respectively. C1, C2, C3, C4 and C5 were located from 1590-2699 nt (369 aa), 1252-1656 nt (134 aa), 1107-1511nt (134 aa), 2010–2324 nt (85 aa) to 319-615 nt (97 aa), respectively. These may putatively encode the replication associated protein (Rep), transcriptional activator protein (TrAP), replication enhancer protein (REn), C4 protein and C5 protein, respectively. The intergenic region (IR) was located from 2700-2844 nt (left IR) to 1-174 nt (right IR) (Fig. 2). The Rep binding site contained a 13 bp element “TTGGG-GAC-TTGGG” which harbored two 5 bp direct repeats separated by a central core of 3 bp, seven nucleotide apart from this repeat a TATA box and 19 nucleotide apart an invert repeat (CCCAA). The stem loop structure contained AGCGGCCAT inverted repeat sequence separated by a nonanucleotide (TAATATTAC) sequence. These features indicated that the genome organization of these isolates was similar to that of DNA-A genome of Old World monopartite begomoviruses. 3.4. Sequence identities BLASTN analysis of begomovirus isolates under study: JI (KC513823), JP (KF652078) and JM (KF652077) revealed 97–98% nucleotide sequence identities with each other and 93–95% identities with an uncharacterized begomovirus of J. curcas (the only sequences available in GenBank database as GU451249 and EU798996). The identities were highest 73% with other begomovirus isolates: catharanthus yellow mosaic virus (CYMV, HE580234) and chili leaf curl Salem virus (CLCSV, HM007119), and chili leaf curl virus (CLCV, JN555600) with more than 82% query coverage. These also showed 79–80% identities with jatropha yellow mosaic India virus (JYMIV; FJ177030) of J. gossypifolia and croton

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249

250

251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282

283

284 285 286 287 288 289 290 291 292 293 294

G Model VIRUS 96547 1–9

ARTICLE IN PRESS A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

4

Fig. 1. Natural symptoms of severe yellow mosaic on J. integerrima (a) and J. podagrica (c), and mild yellow mosaic on J. multifida (e) plants as compared to their respective healthy plants (b, d and f).

295 296 297 298 299 300 301 302 303 304

yellow vein mosaic virus (CYVMV; FN678906) of Alcea rosea (with 67% query coverage). The pairwise sequence analysis of DNA-A genomes of JI, JP and JM isolates along with selected begomoviruses revealed 94–95% identities with each other and 93–95% identities with an uncharacterized begomovirus (GU451249 and EU798996, for which the tentative name given as jatropha leaf curl virus; JLCV). They showed highest 61% identity with JYMIV (FJ177030) and CYVMV (FN678906) reported from India and Pakistan, respectively. The identities were 54–59% with CLCV (HM007119, JN555600 and

HM992939); CYMV (HE580234); pepper yellow leaf curl Lahore virus (PeYLCLV; KC149939, KC149941, KC149940 and KC149938), pepper yellow leaf curl Bangladesh virus (PeYLCBV; HM007097) and CYVMV (EU727086, FN645901 and FN645898) (Table 2). The identities of JI, JP and JM isolates under study at the amino acid residues level of various ORFs were 94–97% for V2, 81–98% for V1, 93–96% for C3, 87–90% for C2, 90–97% for C1 and 86–90% for C4 with two uncharacterized begomovirus isolates (JLCV). The identities were 89% for C3, 70% for C1, 62% for C2, 58% for V1, 50% for V2 and 42% for C4 ORFs with a characterized begomovirus (JYMIV,

Table 1 Whitefly transmission of begomovirus isolates from three Jatropha species and their detection by PCR from inoculated plants after 35 dpi. Begomovirus isolates of Jatropha species

Test plants taken for whitefly transmission

Number of plants with yellow mosaic symptoms/total inoculated plants

Number of positive plants/total tested plants by PCR*

J. integerrima

J. integerrima J. multifida J. podagrica N. glutinosa S. lycopersicum

3/3 2/3 2/3 5/5 5/5

3/3 3/3 3/3 5/5 5/5

J. multifida

J. integerrima J. multifida J. podagrica N. glutinosa S. lycopersicum

2/3 3/3 3/3 5/5 5/5

3/3 3/3 3/3 5/5 5/5

J. podagrica

J. integerrima J. multifida J. podagrica N. glutinosa S. lycopersicum

2/3 2/3 3/3 5/5 5/5

3/3 3/3 3/3 5/5 5/5

*

PCR using begomovirus primers: PALIv 722/PALIc 1960 (Reddy et al., 2005).

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

305 306 307 308 309 310 311 312 313 314

G Model

ARTICLE IN PRESS

VIRUS 96547 1–9

A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

5

Table 2 Pairwise identity matrixes of JM isolate (KF652077) under study at DNA-A genome and their ORFs with respective sequences of JP and JI isolates (under study) and selected begomoviruses using Genomatix DiAlign program. Accession

Abbreviated virus names

Genome size

Host

Locations

DNA-A nt

V2 aa

V1 aa

C3 aa

C2 aa

C1 aa

C4 aa

LIR nt

RIR nt

IR nt

KF652078 GU451249 KC513823 EU798996 FJ177030 FN678906 AM691745 EU727086 FN645898 FN645901 HM007097 KC149939 KC149941 KC149940 KC149938 HE580234 HM007119 JN555600 HM992939

JP isolate JLCV JI isolate JLCV JYMIV CYVMV PeLCLV CYVMV CYVMV CYVMV PeLCBV PeYLCCV PeYLCCV PeYLCCV PeYLCCV CYMV CLCSV CLCV CLCV

2844 2844 2844 2743 2757 2752 2747 2757 2760 2749 2760 2748 2748 2748 2748 2754 2783 2754 2785

Jatropha podagrica J. curcas J. integerrima J. curcas J. gossypifolia Alcea rosea Capsicum annum J. gossypifolia Acalypha sp. Acalypha sp. Chilli Pepper Pepper Pepper Pepper Catharanthus roseus Chilli Chilli Chilli

India India India India India Pakistan Pakistan India Itali India India China China China China Pakistan India India India

95 95 94 93 61 61 60 59 59 59 58 58 58 58 58 57 54 54 54

97 96 94 94 50 52 48 51 50 50 49 50 50 50 50 50 41 34 40

98 – 98 81 58 58 58 53 54 54 58 55 55 55 55 57 57 57 57

95 96 94 93 89 67 63 70 64 64 62 61 61 61 61 61 62 62 26

90 90 88 87 62 35 32 38 34 35 33 38 37 38 37 35 35 36 26

97 – 93 90 70 78 77 79 79 78 77 76 76 76 76 81 76 76 56

90 – – 86 42 71 74 71 74 71 71 65 65 65 65 – 68 67 68

91 91 91 88 53 58 63 66 59 64 56 59 59 59 59 61 60 49 57

89 91 90 92 57 45 51 43 46 46 52 46 43 46 43 45 36 26 37

90 91 90 90 44 52 52 54 52 55 53 51 50 51 50 51 43 33 34

Abbreviations used for this study: CLCV, chilli leaf curl virus; CLCSV, chilli Leaf curl Salem virus; CYMV, catharanthus yellow mosaic virus; CYVMV, croton yellow vein mosaic virus; JLCV, jatropha Leaf curl virus; JYMIV, jatropha yellow mosaic India virus; PeLCBV, pepper leaf curl Bangladesh virus; PeLCLV, pepper leaf curl Lahore virus; PeYLCCV, pepper yellow leaf curl China virus; nt, nucleotide; aa, amino acids; –, data not available.

315 316 317 318 319 320 321 322

FJ177030) isolated from J. gossypifolia in India (Snehi et al., 2011a) (Table 2). However, the degree of identities at complete genome and ORFs levels were very low with other begomoviruses considered for study, suggesting the high genetic variability. These identities were much below than that of the threshold (89% identity) value, the criteria suggested for defining the new begomovirus species (Fauquet et al., 2008; King et al., 2011), therefore, JI, JP and JM isolates, as well as two uncharacterized begomovirus isolates

(tentatively designated as JLCV) were identified as the members of a new species of begomovirus for which the name “Jatropha mosaic Lucknow virus (JMLV)” has been provisionally proposed. The amino acid sequences of V1 (CP) ORF of JI, JP and JM isolates under study were also aligned with the similar sequences of other begomoviruses which showed high variability in the last 45 amino acid residues at C-terminal region (having similar function as nuclear shuttle protein of bipartite begomovirus) from position 201 to 266. The 21 amino acids “FYRVNNYVVYNHQEAAKYENH” present in others begomoviruses were found missing in JI, JP and JM isolates while they contained eight extra amino acids “HQIQCMQH” which were found absent in other begomoviruses (Fig. 3). However, the N-terminal region showed least variability (changes in a few amino acids only) from 1 to 200 amino acid positions. Based on high variability in last 45 amino acid residues of the C-terminal region observed with the begomoviruses taken for study, the JI, JP and JM isolates were considered as members of the distinct begomovirus species. 3.5. Phylogenetic relationships

Fig. 2. Genomic organization of JI (KC513823), JP (KF652078) and JM (KF652077) isolates under study showing arrangement of seven predicted ORFs along with their nucleotide coordinates and putative proteins. Arrows indicate the positions and orientations (virion/complementary sense) of putative ORFs: V2 = pre-coat protein gene, V1 = coat protein gene in virion sense (clock wise), C5 = C5 protein gene, C3 = replication enhancer protein gene, C2 = transcription activator protein gene, C1 = replication associated protein gene and C4 = C4 protein gene in complementary sense (anti clock wise). The intergenic region is represented as filled blocks with a hairpin structure at the origin of replication.

During phylogenetic analysis, the begomovirus isolates: JI (KC513823), JP (KF652078) and JM (KF652077) under study along with two uncharacterized begomovirus isolates (GU451249 and EU798996) grouped together and formed an independent cluster from all other viruses (Fig. 4). Since, the V1 (CP) and C1 (Rep) ORFs are most conserved regions in the begomovirus genome, the phylogenetic trees were also constructed using amino acid sequences of these ORFs of begomovirus under study and other members of family Geminiviridae (selected based on BLASTP) to strengthen the phylogenetic relationships. The phylogenetic analysis using amino acid sequences of V1 (CP) and C1 (Rep) ORFs (Fig. 5a and b, respectively) the isolates under study were located on a discrete branch, demonstrating the virus to be a distinct and highly divergent begomovirus species. 3.6. Recombination analysis of begomoviral genome RDP analysis did not show any possible recombination breakpoint in genome of begomovirus isolates: JI (KC513823), JP (KF652078) and JM (KF652077) under study suggesting that they

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340

341

342 343 344 345 346 347 348 349 350 351 352 353 354 355

356

357 358 359

G Model VIRUS 96547 1–9 6

ARTICLE IN PRESS A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

Fig. 3. Alignment of V1 (coat protein) ORF of JI, JP and JM isolates under study with other begomoviruses showing high variability in 45 amino acid residues of the C-terminal region. The highly variable region has been shown in the box. # = isolates under study, - = amino acid sequences almost similar, * = deletions/missing amino acid sequences.

Fig. 4. Phylogenetic relationships of JI (KC513823), JP (KF652078) and JM (KF652077) isolates under study with respective sequence data of selected begomoviruses reported worldwide. Phylogenetic analysis was performed by MEGA v6.0 with 1000 replicates bootstrapping and the dendrogram was generated with NJ-method and viewed by NJ plot program. The pairwise identities of begomovirus isolates under study with other begomoviruses are also shown.

Fig. 5. Phylogenetic relationships of amino acid sequences of coat protein (a) and Rep (b) of JI, JP and JM isolates under study with some selected begomoviruses and other members of family Geminiviridae (based on BLASTP analysis) generated using MEGA v6.0 with 1000 replicates bootstrapping. The dendrogram was generated with Maximum likelyhood-method and viewed by ML plot program.

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

G Model VIRUS 96547 1–9

ARTICLE IN PRESS A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

7

Fig. 6. Similarity plot analysis of nucleotide sequence of genome of begomovirus isolates: JI, JP and JM (under study) was performed using Simplot program, version 3.2, with a sliding window of 200 nucleotides moving in 20-nucleotide steps. The green, red, yellow, blue and gray plots represent JLCV-Guj, CYVMV, CYMV, PLCLV and CLCV isolates. The maximum and minimum bounds of the scans represent the degrees of sequence similarity expected following alignment amongst unrelated sequences with Q9 the same nucleotide composition as the real begomovirus sequences. The linear presentation of begomoviral genome has also been shown on X-axis. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)

360 361 362 363 364 365 366 367 368

369

370 371 372 373 374 375 376 377 378 379 380 381

did not evolve by recombination. Simplot analysis also supported the findings of RDP. The Simplot analysis clustered the begomovirus isolates under study into a single group and other begomoviruses into different groups which indicated that the begomovirus isolates under study were highly dissimilar with other begomoviruses. The analysis also revealed least similarity of the begomovirus isolates under study at IR, V2, C2, C3 and C4 regions (Fig. 6). These results indicated that the begomovirus isolates under study comprised a new species which has not been characterized previously. 3.7. Agroinfectivity assay The agroinoculated J. integerrima, J. podagrica and J. multifida plants with pCAM-JM-A developed yellow mosaic symptoms at 35 dpi, which were similar to that of naturally infected plants, proving the Koch’s postulates, but no symptoms were developed on these plants when agroinoculated with mock (pCAM) (Table 3). S. lycopersicum, N. glutinosa and N. benthamiana plants developed yellow mosaic and leaf curl symptoms on newly emerging leaves but mock inoculated plants did not show any symptoms. The PCR analysis resulted in the positive band of expected size (∼1.2 kb) in all inoculated plants with pCAM-JM-A but not in any of the mock inoculated plants (Table 3), confirming the presence of begomovirus in all inoculated plants with pCAM-JM-A. The infectivity

assay indicated that these isolates were similar to that of DNA-A genome of Old World monopartite begomoviruses. 4. Discussion In this study, the causal agent of yellow mosaic disease of three important ornamental Jatropha species: J. integerrima, J. podagrica and J. multifida grown in Indian gardens was transmitted by whitefly and Koch’s postulates were established by inoculations of agroinfectious clones. The causal virus was identified as a new begomovirus species on the basis of low nucleotide sequence identities [(which were much below than 89%, the threshold value of species demarcation criteria for a new begomovirus species proposed by Faquet et al. (2008) and King et al. (2011)] and Q7 highly distant phylogenetic relationships of the complete genome sequences with respective sequences of the other begomoviruses available in GenBank database. The genome of begomovirus under study contained 6 potential ORFs: V2 (pre-coat protein) and V1 (coat protein) in viron sense and C1 (replication associated protein), C2 (transcription activator protein), C3 (replication enhancer protein) and C4 (C4 protein) in complementary sense strands. However, the presence of C5 ORF (C5 protein) was a unique feature of begomovirus under study which has been found in a few monopartite begomoviruses and suggested that the C5 gene

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

382 383

384

385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403

G Model

ARTICLE IN PRESS

VIRUS 96547 1–9

A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

8

Table 3 Infectivity of agroinfectious clone of begomovirus isolate (JM-A) on three host plants. Species infiltrated

Agroinfectious clones

Symptoms observed (at 35 dpi)

Number of symptomatic/inoculated plants

Detection of DNA genome by PCR*

Jatropha multifida

pCAM-JM-A Mock (pCAM)

Yellow mosaic No symptoms

3/3 0/3

3/3 0/3

J. integerrima

pCAM-JM-A Mock (pCAM)

Yellow mosaic No symptoms

3/3 0/3

3/3 0/3

J. podagrica

pCAM-JM-A Mock (pCAM)

Yellow mosaic No symptoms

3/3 0/3

3/3 0/3

Nicotiana glutinosa

pCAM-JM-A Mock (pCAM)

Yellow mosaic and leaf curl No symptoms

3/3 0/3

3/3 0/3

N. benthamiana

pCAM-JM-A Mock (pCAM)

Yellow mosaic and leaf curl No symptoms

3/3 0/3

3/3 0/3

Solanum lycopersicum

pCAM-JM-A Mock (pCAM)

Yellow mosaic and leaf curl No symptoms

3/3 0/3

3/3 0/3

*

404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448

PCRs were done using begomovirus universal primers (Reddy et al., 2005).

had either no effect on viral infectivity and symptom development or induced very mild leaf curl and light green mosaic across the leaf lamina (Amin et al., 2011; Melgarejo et al., 2013). The Rep binding site of IR contained a 13 bp iterated sequences, a TATA box and an inverted repeat sequence needed for the rolling circle replication of begomovirus (Akbar-Behjatnia et al., 1998). The stem loop structure contained AGCGGCCAT inverted repeat sequence separated by a nonanucleotide (TAATATTAC) sequence needed for replication of begomovirus (Akbar-Behjatnia et al., 1998). These features suggested that the genome organization of these isolates was similar to that of DNA-A genome of Old World monopartite begomoviruses reported worldwide (Rybicki, 1994). Since, begomoviruses have evolved rapidly through changes in their DNA genome, the best adapted mutants and intra/inter genus recombination for evolving a new species of begomovirus has been suggested earlier (Monjane et al., 2014), therefore, the possibility of recombination has also been investigated by RDP and similarity plot analyses. The analyses suggested that begomovirus under study has not been evolved by recombination with any known begomovirus which was not in accordance with the earlier study of Monjane et al. (2014). It suggested an independent evolution of this species. Moreover, the phylogenetic analysis also supported the RDP analysis which showed that begomovirus under study did not cluster any begomovirus, hence, was considered as a distinct begomovirus species. A literature survey revealed reports of two begomoviruses: ACMV from Kenya and Jatropha mosaic virus from Florida which can cause a mosaic disease of J. multifida (Bock et al., 1981; Polston et al., 2014). The occurrence of begomoviruses has also been reported on J. curcas (Aswatha-Narayana et al., 2006, 2007; Tewari et al., 2007; Raj et al., 2008; Gao et al., 2010; Ramkat et al., 2011a,b; Snehi et al., 2012) and J. gossypifolia (Snehi et al., 2011a,b). Two sequences of uncharacterized begomovirus isolates (tentatively named as JLCV) from J. curcas are also available in GenBank database. However, the occurrence of neither begomovirus nor any other virus has been reported so far on J. integerrima and J. podagrica. The virus under study can infect Euphorbiaceae as well as Solanaceae and can therefore spread from ornamental Jatropha hosts to important crop plants such as tomato. The three Jatropha species are therefore newly identified reservoir plants. Here we report the characterization of a new begomovirus species associated with yellow mosaic disease of J. integerrima, J. podagrica and J. multifida for the first time for which the name “Jatropha mosaic Lucknow virus” has been proposed.

5. Conclusions

449

A begomovirus associated with yellow mosaic disease of J. integerrima, J. podagrica and J. multifida was shown to be an “Old World” monopartite begomovirus, based on the sequence analyses of the cloned ∼2.9 kb genome. It shared 61% genome sequence identity and a distant phylogenetic relationship with other begomoviruses reported worldwide and hence is proposed to be a new species of begomovirus. Uncited references Briddon et al. (2001). Acknowledgements Authors are thankful to Director, CSIR-NBRI for facilities and Department of Biotechnology (DBT) for funding under the project GAP-246425. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.virusres.2015.02.015. References Akbar-Behjatnia, S.A., Dry, I.B., Rezaian, M.A., 1998. Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus. Nucl. Acids Res. 26 (4), 925–931. Amin, I., Patil, B.L., Briddon, R.W., Mansoor, S., Fauquet, C.M., 2011. Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs. Virol. J. 19 (8), 238. Appiah, A.S., Amoatey, H.M., Klu, G.Y.P., Afful, N.T., Azu, E., Owusu, G.K., 2012. Spread of African cassava mosaic virus from cassava (Manihot esculenta Crantz) to physic nut (Jatropha curcas L.) in Ghana. J. Phytol. 3 (12), 35–40. Aswatha-Narayana, D.S., Shankarappa, K.S., Govindrappa, M.R., Prameela, H.A., GururajRao, M.R., Rangaswamy, K.T., 2006. Natural occurrence of Jatropha mosaic virus disease in India. Curr. Sci. 91, 584–586. Aswatha-Narayana, D.S., Rangaswamy, K.T., Shankarappa, K.S., Maruthi, M.N., Reddy Laksminarayana, C.N., Rekha, A.R., Murthy-Keahava, K.V., 2007. District begomoviruses closely related to cassava mosaic viruses cause Indian Jatropha mosaic disease. Int. J. Virol. 3, 1–11. Bock, K.R., Guthrie, E.J., Figueiredo, G., 1981. A strain of Cassava latent virus occurring in coastal districts of Kenya. Ann. Appl. Biol. 99 (2), 151–159. Briddon, R., Mansoor, S., Bedford, I., Pinner, M., Saunders, K., Stanley, J., Zafar, Y., Malik, K., Markham, P., 2001. Identification of DNA components required for induction of Cotton leaf curl disease. Virology 285, 234–243.

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

450 451 452 453 454 455 456

Q8

457

458

459

460 461 462

463

464 465 466

467

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489

G Model VIRUS 96547 1–9

ARTICLE IN PRESS A. Srivastava et al. / Virus Research xxx (2015) xxx–xxx

490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547

Briddon, R.W., Bull, S.E., Amin, I., Idris, A.M., Mansoor, S., Bedford, I.D., Dhawan, P., Rishi, N., Siwatch, S.S., Abdel-Salam, A.M., Brown, J.K., Zafar, Y., Markham, P.G., 2003. Diversity of DNA ␤: a satellite molecule associated with some monopartite begomoviruses. Virology 312, 106–121. Dehgan, B., 1982. Novel Jatropha for Florida Landscapes, vol. 95. Proceedings of the Florida State Horticultural Society, pp. 277–280. Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1 (4), 19–21. Fauquet, C.M., Briddon, R.W., Brown, J.K., Moriones, E., Stanley, J., Zerbini, M., Zhou, X., 2008. Geminivirus strain demarcation and nomenclature. Arch. Virol. 153, 783–821. Gao, S., Qu, J., Chua, N.H., Ye, J., 2010. A new strain of Indian cassava mosaic virus causes a mosaic disease in the biodiesel crop Jatropha curcas. Arch. Virol. 155, 607–612. Hanley-Bowdoin, L., Bejarano, E.R., Robertson, D., Mansoor, S., 2013. Geminiviruses: masters at redirecting and reprogramming plant processes. Nat. Rev. 11, 777–788. Hooker, W.J., Prain, D., Stapf, O., 1848. Curtis Botanical Magazine. Royal Botanic Gardens, Kew, pp. 4376–4379. Jeske, H., 2009. Geminiviruses. Curr. Top. Microbiol. Immunol. 331, 185–226. Kashina, B.D., Alegbejo, M.D., Banwo, O.O., Nielsen, S.L., Nicolaisen, M., 2013. Molecular identification of a new begomovirus associated with mosaic disease of Jatropha curcas L. in Nigeria. Arch. Virol. 158 (2), 511–514. King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), 2011. Virus Taxonomy-Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press, London, United Kingdom. Kirtikar, K.R., Basu, B.D., 2002. Indian Medicinal Plants. Popular Prakashan, Dehradun, pp. 1570–1579. Kon, T., Gilbertson, R.L., 2012. Two genetically related begomoviruses causing tomato leaf curl disease in Togo and Nigeria differ in virulence and host range but do not require a betasatellite for induction of disease symptoms. Arch. Virol. 157 (1), 107–120. Martin, D.P., Lemey, P., Lott, M., Moulton, V., Posada, D., Lefeuvre, P., 2010. RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26, 2462–2463. Melgarejo, T.A., Kon, T., Rojas, M.R., Paz-Carrasco, L., Zerbini, F.M., Gilbertson, R.L., 2013. Characterization of a new world monopartite begomovirus causing leaf curl disease of tomato in Ecuador and Peru reveals a new direction in geminivirus evolution. J. Virol. 87 (10), 5397–5413. Monjane, A.L., Martin, D.P., Lakay, F., Muhire, B.M., Pande, D., Varsani, A., Harkins, G., Shepherd, D.N., Rybicki, E.P., 2014. Extensive recombination-induced disruption of genetic interactions is highly deleterious but can be partially reversed by small numbers of secondary-recombination events. J. Virol., http://dx.doi.org/10.1128/JVI. 00709-14. Morgenstern, B., Frech, K., Dress, A., Werner, T., 1998. DIALIGN: finding local similarities by multiple sequence alignment. Bioinformatics 14, 290–294. Nayak, B.S., Patel, K.N., 2009. Designing and characterization of drug free patches for transdermal application. Int. J. Pharm. Res. 1 (3), 35–39. Ojewole, J.A.O., Odebiyi, O.O., 1980. Neuromuscular and cardiovascular actions of tetramethylpyrazine from the stem of Jatropha podagrica. Planta Med. 38 (4), 332–338. Openshaw, K., 2000. A review of Jatropha curcas: an oil plant of unfulfilled promise. Biomass Bioenergy 19, 1–15. Padidam, M., Beachy, R.N., Fauquet, C.M., 1995. Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. J. Gen. Virol. 76, 25–35. Polston, J.E., London, M.A., Capobianco, H., 2014. The complete genome sequence of New World Jatropha mosaic virus. Arch. Virol., http://dx.doi.org/10.1007/ s00705-014-2132-1.

9

Pramanik, K., 2003. Properties and use of Jatropha curcas oil and diesel fuel blends in compression ignition engine. Renew. Energy 28, 239–248. Raj, S.K., Snehi, S.K., Kumar, S., Khan, M.S., Pathre, U., 2008. First molecular identification of a begomovirus in India that is closely related to Cassava mosaic virus and causes mosaic and stunting of Jatropha curcas L. Australas. Plant Dis. Notes 3, 69–71. Ramkat, R.C., Alberto, C., Fatemeh, M., Margit, L., 2011a. Biotechnological approaches to determine the impact of viruses in the energy crop plant Jatropha curcas. Virol. J. 8, 386. Ramkat, R.C., Alberto, C., Fatemeh, M., Margit, L., 2011b. Occurrence of African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda (EACMV-UG) in Jatropha curcas. BMC Proc. 5, P93. Reddy, R.V.C., Colvin, J., Muniyappa, V., Seal, S., 2005. Diversity and distribution of begomoviruses infecting tomato in India. Arch. Virol. 150, 845–867. Rybicki, E.P., 1994. A phylogenetic and evolutionary justification for three genera of Geminiviridae. Arch. Virol. 139 (1–2), 49–77. Sambrook, J., Russel, D.W., 1989. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, pp. 213p. Saunders, K., Bedford, I.D., Briddon, R.W., Markham, P.G., Wong, S.M., Stanley, J., 2000. A novel virus complex causes Ageratum yellow vein disease. Proc. Natl. Acad. Sci. U. S. A. 97, 6890–6895. Sharma, S.K., Singh, H., 2013. Pharmacognostical standardisation of Jatropha integerrima Jacq. (Euphorbiaceae) roots. Der Pharmacia Lettre 5 (1), 155–159. Simmonds-Gordon, R.N., Collins-Fairclough, A.M., Stewart, C.S., Roye, M.E., 2014. First report of a complete genome sequence for a begomovirus infecting Jatropha gossypifolia in the Americas. Arch. Virol., http://dx.doi.org/10.1007/ s00705-014-2112-5. Snehi, S.K., Khan, M.S., Raj, S.K., Prasad, V., 2011a. Complete nucleotide sequence of Croton yellow vein mosaic virus and DNA-␤ associated with yellow vein mosaic disease of Jatropha gossypifolia in India. Virus Genes 43, 93–101. Snehi, S.K., Raj, S.K., Khan, M.S., Prasad, V., 2011b. Molecular identification of a new begomovirus associated with yellow mosaic disease of Jatropha gossypifolia in India. Arch. Virol. 156, 2303–2307. Snehi, S.K., Srivastava, A., Raj, S.K., 2012. Biological characterization and complete genome sequence of a possible strain of Indian cassava mosaic virus from Jatropha curcas in India. J. Phytopathol. 160, 547–553. Speight, J.G., Singh, K., 2014. Environmental Management of Energy From Biofuels and Biofeedstocks. John Wiley & Sons, http://dx.doi.org/10.1002/ 9781118915141. Srivastava, A., Raj, S.K., Kumar, S., Snehi, S.K., Kulshreshtha, A., Hallan, V., Pande, S.S., 2013. Molecular identification of Ageratum enation virus, betasatellite and alphasatellite molecules isolated from yellow vein diseased Amaranthus cruentus in India. Virus Genes 47 (3), 584–590. Srivastava, A., Kumar, S., Raj, S.K., Pande, S.S., 2014a. Association of a distinct strain of Hollyhock yellow vein mosaic virus and ludwigia leaf distortion betasatellite with yellow vein mosaic disease of hollyhock (Alcea rosea) in India. Arch. Virol. 159 (10), 2711–2715. Srivastava, A., Jaidi, M., Kumar, S., Raj, S.K., 2014b. Molecular identification of a new begomovirus associated with leaf crumple disease of Jatropha curcas L. in India. Arch. Virol., http://dx.doi.org/10.1007/s00705-014-2288-8. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tewari, J.P., Dwivedi, H.D., Pathak, M., Srivastava, S.K., 2007. Incidence of a mosaic disease in Jatropha curcas L. from eastern Uttar Pradesh. Curr. Sci. 93, 1048–1049. Tiwari, S.P., Nema, S., Khare, M.N., 2013. Whitefly – a strong transmitter of plant viruses. eSci J. Plant Pathol. 2 (2), 102–120. Zhang, H., Ma, X.Y., Qian, Y.J., Zhou, X.P., 2010. Molecular characterization and infectivity of Papaya leaf curl China virus infecting tomato in China. J. Zhejiang Univ. Sci. 11 (2), 109–114.

Please cite this article in press as: Srivastava, A., et al., Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.02.015

548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606