Molecular identification of a Cucumber mosaic virus ... - Springer Link

Journal of Plant Diseases and Protection, 116 (5), 193–199, 2009, ISSN 1861-3829. © Eugen Ulmer KG, Stuttgart

Molecular identification of a Cucumber mosaic virus subgroup II isolate from carrot (Daucus carota) based on RNA3 genome sequence analyses Molekulare Identifizierung eines Isolats des Gurkenmosaikvirus (Untergruppe II) aus der Möhre (Daucus carota) mittels Sequenzanalyse des RNA-3-Genoms B. Afreen1, A.A. Khan1, Q.A. Naqvi1, S. Kumar2, D. Pratap2, S.K. Snehi2 & S.K. Raj2,* Department of Botany, Aligarh Muslim University, Aligarh, India Plant Molecular Virology, National Botanical Research Institute, Lucknow, India Corresponding author, e-mail [email protected]

1 2 *

Received 16 March 2009; accepted 16 July 2009

Abstract Natural occurrence of severe chlorotic mottle disease of carrot (Daucus carota) with a significant incidence was observed in northern Uttar Pradesh, India. The causal pathogen was successfully transmitted to a number of test plants through sap inoculations and by aphid (Aphis gossypii Glover). The association of Cucumber mosaic virus (CMV) was detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by sequencing of ∼2.2 Kb amplicons. Analyses of sequence data (EU642567) revealed the presence of 2203 nucleotides of complete RNA3 which included two open reading frames (ORFs): movement protein (840 nucleotides translating 279 amino acid residues) and coat protein (657 nucleotides translating 218 amino acid residues). During BLAST analysis of complete RNA3 sequence, carrot virus isolate shared highest 97% identities with several strains of CMV of subgroup II reported worldwide. Phylogenetic analysis using RNA3 sequence data of selected strains of CMV of subgroup IA, IB and II showed the close relationship of carrot isolate with the strains of CMV of subgroup II. Based on highest sequence identities (97%) and close phylogenetic relationship, the causal virus of mottle disease in carrot was identified as an isolate of CMV belonging to subgroup II. The association of CMV of subgroup II with chlorotic mottle disease of D. carota is a first report from India. Key words: carrot, Cucumber mosaic virus, sequence analyses of RNA3, subgroup II

Zusammenfassung Im Norden des indischen Bundesstaates Uttar Pradesh wurden höhere Befallshäufigkeiten einer chlorotischen Sprenkelkrankheit der Möhre (Daucus carota) beobachtet. Der Erreger konnte durch mechanische Saftinokulation oder durch Blattläuse (Aphis gossypii Glover) auf verschiedene Testpflanzen übertragen werden. Die Reverse-Transkriptase-Polymerasekettenreaktion (RT-PCR) zeigte eine Beteiligung des Gurkenmosaikvirus (CMV) an der Möhrenkrankheit, was durch die Sequenzierung eines ∼2.2 Kb großen Amplicons bestätigt wurde. Die Sequenzanalyse des Amplicons (EU642567) ergab 2203 Nukleotide der vollständigen RNA3 des CMV mit zwei offenen Leserahmen (ORFs), dem Transportprotein (840 Nukleotide, die 279 Aminosäuren kodieren) und dem Hüllprotein (657 Nukleotide und 218 Aminosäuren). Während der BLAST-Analyse der vollständigen RNA3-Sequenz zeigte das Virusisolat aus der Möhre die höchste Übereinstimmung (97%) mit verschiedenen Stämmen der Untergruppe II des CMV aus allen Erdteilen. Eine phylogenetische Analyse von RNA3-Sequenzdaten ausgewählter CMV-Stämme der Untergruppen IA, IB und II bestätigte die enge Beziehung zwischen dem indischen Möhrenisolat und den CMV-Stämmen der J.Plant Dis.Protect. 5/2009

Untergruppe II. Aufgrund der 97%igen Sequenzübereinstimmung und der engen phylogenetischen Beziehung wurde der Erreger der chlorotischen Sprenkelkrankheit der Möhre als CMVIsolat der Untergruppe II identifiziert. Es handelt sich um die erste Beschreibung des CMV der Untergruppe II als Erreger der chlorotischen Sprenkelkrankheit der Möhre in Indien. Stichwörter: Gurkenmosaikvirus, Möhre, Sequenzanalyse der RNA3, Untergruppe II

1

Introduction

Carrot (Daucus carota, family Apiaceae) is one of the major consuming food crops grown all over the world because of its high nutritional value, especially for vitamin A. In India the crop is being grown commercially round the year in the area of 20,124 ha with an annual production of 28,70,007 tons (SIDHU 1998). Northern Uttar Pradesh (U.P.) contributes the significant production of the total carrot productivity in India. During a survey in November-December, 2007 a number of carrot plants growing in the fields at Aligarh, U.P. (India) were found to be exhibiting severe chlorotic mottle disease symptoms with significant disease incidence. Literature survey revealed the reports on natural occurrence of several viruses on carrot from all over the world viz. Carrot mottle dwarf virus (STUBBS 1952); Carrot thin leaf potyvirus (HOWELL and MINK 1976); Alfalfa mosaic virus, Carrot latent virus, Carrot mottle virus, Carrot red leaf virus, Carrot yellow leaf virus, Celery mosaic virus, Curly top virus (MURANT et al. 1985) and Carrot virus Y (LATHAM and JONES 2004) which consequently cause tremendous yield reduction and its quality production. However, any report of Cucumber mosaic virus (CMV) infection on carrot is not available in literature from any part of the world. We report the natural occurrence of an isolate of CMV of subgroup II on carrot (D. carota) for the first time. CMV of genus Cucumovirus, family Bromoviridae has great economic importance because of its largest host range among known plant viruses, which is reported to infect approximately 1000 plant species representing 86 families including both monocots and dicots (PALUKAITIS and GARCIA-ARENAL 2003). The genome of CMV consists of three single-stranded positive-sense RNA species designated as RNA1, RNA2, and RNA3. RNA1 and RNA2 encode Ia and 2a proteins, respectively, essential for replication. RNA3 is dicistronic and encodes the movement protein (MP) and coat protein (CP). MP encoded by an ORF located within the 5’-half of RNA3 is involved in cell-to-cell movement of the virus. The second ORF located within the 3’-half encodes the 24 kDa CP is expressed through a sub-genomic RNA4. The CP has been demonstrated to have role in encapsidation, systemic movement within infected plants, host range and aphid transmission (PALUKAITIS and GARCIA-ARENAL 2003). The strains of CMV have been divided into two subgroups (I and II) based on serological

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Afreen et al.: Molecular identification of a Cucumber mosaic virus subgroup II isolate from carrot

properties, nucleic acid hybridization, nucleic acid and/or protein sequence composition, RNase protection assay and RT-PCR RFLP analysis. Further splitting of subgroup I into IA and IB has been proposed on the basis of sequence data, analysis of 5’-non-translated region of RNA3 of several strains and phylogenetic analysis of CP (ROOSSINCK 2002) and accordingly the Asian strains including the Indian ones have been grouped into the subgroup IB (PALUKAITIS and GARCIA-ARENAL 2003). Our study represents the biological and molecular identification of CMV of subgroup II isolated from carrot based on sequence analysis of RNA3 genome.

25 pM each of forward and reverse primers and 3 U Pfu DNA polymerase (MBI Fermentas) in a Peltier thermal cycler PTC200 engine (MJ Research, Waltham, MA, USA). The PCR conditions were: initial template denaturation at 94°C for 5 min was followed by 25 cycles consisting of denaturation at 94°C for 1 min, primers annealing at 52°C for 45 s and extension at 72°C for 3 min and a final extension at 72°C for 10 min. The PCR products were assessed on 1% agarose gel with DNA marker (lambda DNA digested with EcoRI/HindIII, Genei, Bangalore, India).

2.4 Cloning and sequencing of PCR amplicons 2 Materials and methods 2.1 Virus culture and virus transmission by mechanical inoculations The leaf samples from naturally infected carrot (D. carota) plants showing severe chlorotic mottle symptoms were collected from the fields nearby Aligarh (U.P.), India. These samples were used for all experimental work. For mechanical inoculation tests, the naturally infected young leaves of carrot were macerated in 0.1M potassium phosphate buffer, pH 6.8 (supplemented with 10 mM ethylene diamine tetra acetic acid and 0.1% sodium sulphite) in a ratio of 1:10 (w/v) using a sterile pestle and mortar. The homogenate obtained after squeezing through double layered muslin cloth was rubbed onto carborandum pre-dusted leaves of various host species. The test species viz. Chenopodium amaranticolor, C. album, Cucumis sativus, Datura metal, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. tabacum cv. Samsun NN, N. tabacum cv. White Burley and Vigna unguiculata were inoculated and observed for one month for symptom development, if any.

2.2 Virus transmission through aphids For aphid transmission studies, healthy aphids (Aphis gossypii Glover, family Aphididae) reared on healthy N. tabacum cv. White Burley were starved for 2 hrs and allowed for acquisition access on naturally infected carrot leaf for 2–3 min. The aphids were then transferred to six healthy N. tabacum cv. White Burley plants (10 aphids/plant) for inoculation access of 2 hrs and then were killed by spraying 0.1% Confidor insecticide (Bayer Crop Science Ltd., Mumbai, India). The inoculated plants were kept in an insect proof cage and observed for six weeks for the symptom development, if any.

The PCR amplicons of ∼2.2 kb were eluted through Wizard SV Gel and PCR clean-up system (Promega, Madison, WI, USA) and ligated into pGEM-T Easy vector system (Promega) as per the manufacturer’s instructions. The competent Escherichia coli cells (strain DH5α) were transformed and clones were selected on Luria agar plates supplemented with ampicillin (100 µg ml–1). The presence of insert was checked through digestion of clones by EcoRI restriction enzyme. Three positive clones were got sequenced and consensus sequence data obtained was deposited in GenBank database.

2.5 Sequence analysis RNA3 sequence data were analyzed by BLAST (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) and compared with existing sequences of CMV strains available in GenBank database. The matrix for pair wise alignment of selected CMV strains (obtained from BLAST) was carried out by Genomatix DiAlign version 2 (MORGENSTERN 1999). ORF in sequence data was predicted by ORF finder (www.ncbi.nlm.nih.gov/projects/gorf/) to find out in frame AUG-start and TAG-termination codons. ORFs were translated into amino acid residues using Expasy tool (www.expasy.org/tools/dna.html). Phylogenetic analyses were perused by MEGA tool version 4 (TAMURA et al. 2007). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches and phylogenetic tree was generated with neighbour-joining method and viewed by the NJplot program. All positions containing gaps and missing data were eliminated from the dataset through ‘Complete deletion option’. Peanut stunt virus (PSV) and Tomato aspermy virus (TAV) were considered as referenced out-group members of the genus Cucumovirus for rooting of phylogenetic tree.

3

Results

2.3 RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)

3.1 Symptomatology and virus transmission

The total RNA was extracted from naturally infected and healthy leaf samples of carrot using the RNeasy plant RNA isolation kit (Qiagen, Hilden, Germany) and suspended in 50 µl RNAse-free sterile water and used as templates for RT-PCR. The RT-PCR was performed by using a set of primers capable of amplifying complete RNA3 genome of CMV designed earlier (KUMAR 2008): 5’-GTTTACCAACCAACCAACCACTACT-3’ (forward primer) and 5’-GACCTACGGAAAAACCGTAGGCGAT-3’ (reverse primer). Complementary DNA (cDNA) synthesis was carried out in a 20 µl reaction mixture containing: 1.0 µg of total RNA template, 1 × reverse transcriptase buffer, 1.0 mM each dNTPs, 100 pM reverse primer, 25 U RNA guard (MBI Fermentas, Hanover, MD, USA) and 200 U of MMuLV Reverse transcriptase Revert Aid H-minus (MBI Fermentas). The reaction was performed at 42°C for 90 min. PCR was performed in a 50 µl reaction mixture containing: 50 ng cDNA template, 1 × Pfu DNA polymerase buffer, 200 µM dNTPs, 25 mM MgCl2,

The survey carried out during November–December, 2007 and the natural occurrence of severe chlorotic mottle disease of carrot was observed with disease incidence of 18.7% (28/150). Naturally infected carrot plants exhibited severe chlorosis on young leaves, mottle symptoms on mature leaves, stunting of whole plant (Fig. 1) and deformation in edible root as compared to the healthy plants. Mechanical sap inoculations revealed successful transmission of the virus from carrot to a number of test species. Virus induced necrotic local lesions on C. amaranticolor (Fig. 2a), C. album and N. tabacum cv. White Burley (Fig. 2b) on 7–10 days post inoculations (dpi). The virus also induced systemic chlorosis on V. unguiculata (Fig. 2c), mottling on N. tabacum cv. White Burley (Fig. 2d), D. metal (Fig. 2e), N. benthamiana and N. clevalandii and severe mosaic on C. sativus (Fig. 2f) on 22–25 dpi which is a characteristic feature of CMV. However, inoculated N. glutinosa and N. tabacum cv. Samsun NN neither developed any local nor systemic symptoms even on 30 dpi. J.Plant Dis.Protect. 5/2009


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The virus could also be successfully transmitted through A. gossypii in non-persistent manner from carrot to N. tabacum cv. White Burley. The 3/6 inoculated N. tabacum cv. White Burley plants developed systemic mosaic and mottle symptoms on 25 dpi and also gave local lesions after back inoculation on C. amaranticolor indicating the presence of virus.

3.2 RT-PCR, Cloning and sequencing of PCR amplicons Total RNA isolated from the leaves of naturally infected and healthy carrot plants was subjected to RT-PCR using CMVRNA3 specific primers which resulted in the expected size amplicons of ∼2.2 kb in symptomatic (2/2) samples (Fig. 3), but no such amplicon was obtained in healthy (1/1) sample collected from the same location. The ∼2.2 kb amplicons obtained were successfully cloned in the pGEM-T Easy vector and confirmed by restriction digestion of plasmid DNA isolated from the antibiotic resistant clones indicated presence of insert of the same size. Three such positive clones were sequenced and the consensus sequence data obtained was deposited in GenBank database under the Accession No. EU642567.

3.3 Analysis of sequence data

Fig. 1: Naturally infected carrot plant showing severe mottling in mature leaf and chlorosis in young leaf.

Back inoculation tests of these test plants were also found negative which indicated that these hosts were non host of the virus isolate.

a

d J.Plant Dis.Protect. 5/2009

b

e

Analysis of sequence data (EU642567) revealed the presence of complete CMV-RNA3 of 2203 nucleotides which included two ORFs. The ORF at 5’ end of the sequence was MP gene of 840 nucleotides translating 279 amino acid residues and the ORF at 3’ end was CP gene of 657 nucleotides translating 218 amino acid residues. During BLAST analysis, virus isolate (EU642567) shared highest 97% sequence identities with several strains of CMV of subgroup II reported worldwide (AF198103, Z12818, AB176847, AB006813, L15336, AB189917, AJ304399, EF202597, AF127976 and M21464). Genomatix DiAlign based analysis of complete RNA3 sequence of the virus isolate was also performed with selected

c

f

Fig. 2: Necrotic local lesions obtained on Chenopodium amaranticolor (a), Nicotiana tabacum cv. White Burley (b) after 7–10 dpi of virus inoculation and systemic chlorosis on Vigna unguiculata (c), systemic mosaic and mottle on N. tabacum cv. White Burley (d), Datura metal (e) and Cucumis sativus (f) after 20–25 dpi.

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Afreen et al.: Molecular identification of a Cucumber mosaic virus subgroup II isolate from carrot M

1

2

3

bp 21226 5148 4268 3530 2027 1904

~2.2 Kb

Fig. 3: Agorose gel electrophoresis showing ∼2.2 Kb amplicons during RT-PCR using CMV-RNA3 specific primers in naturally infected carrot samples (1-2), but not in healthy one (3). M: λ DNA/EcoRI/HindIII.

CMV strains of CMV subgroup II, IA and IB (the representatives each of subgroup). The isolate under study revealed 96– 93% sequence identities with CMV strains of subgroup II,

however, the identities with CMV subgroup IA and IB were 63–61% (Table 1). The analysis of CP gene showed highest 96–93% nucleotide identities and 94–90% amino acid identities with several strains of subgroup II reported from India and abroad. The identities were low as 79–76% with CMV stains of subgroup IA and IB (Table 1). The analysis of MP gene also showed highest 98–96% identities at nucleotide and 95–93% at amino acid level with subgroup II members (Table 1). Multiple sequence alignment of CP amino acid residues revealed the presence of 13 unique amino acid substitutions at the positions: 32 M/L, 49 D/G, 65 A/K, 69 P/T, 79 G/E, 176 E/D, 181 S/R, 184 T/A, 188 K/Y, 189 Q/S, 193 N/K, 199 M/I and 201 F/L (Fig. 4). It has also been noticed that there were more substitutions at C-terminus as compared to N-terminus. The two regions at 1–31 and 80–176 amino acid positions were found most conserved (shown by blocks in Fig. 4). Complete RNA3 sequence based phylogenetic analysis clearly grouped the virus isolate with CMV strains of subgroup II reported all over the world, while the members of CMV subgroup IA, IB formed the separate clusters (Fig. 5a). The phylogenetic analysis of amino acid reduces of CP of the virus isolate also revealed a close relationship with the members of CMV subgroup II, reported from abroad, while other Indian strains of subgroup II were found to be distantly related to the carrot isolate (Fig. 5b). Based on highest identities (> 96%) at RNA3 and close phylogenetic relationships with subgroup II members of CMV, the isolate under study was identified as an isolate of CMV of subgroup II and designated as CMV-carrot.

Table 1: Nucleotide (nt) and amino acid (aa) identities of carrot isolate (EU642567) with the selected strains of CMV of subgroup IA, 1B, II and other members of the genus Cucumovirus

Accession

Strains, abbreviated

Natural host

Country

Z12818 AB176847 AF198103 AB006813 AJ304399 L15336 AB189917 EF202597 AF127976 M21464 Y18138 AJ585519 AF268598 EU665002 AJ866272 EU600216 AM396983 AJ585086 U20668 AM114273 Y16926 EF593023 D28780 EU163411 NC_002040

CMV-Kin CMV-TN CMV-LY CMV-M2 CMV-ALS CMV-Trk7 CMV-MT CMV-Tsh CMV-LS CMV-Q CMV-R CMV- 0241 CMV-Xb CMV-TE CMV-Ger CMV-Oci CMV-Teg CMV-Lil CMV-Fny CMV-LeO2 CMV-Tfn CMV-A CMV-Nt9 TAV-Luc PSV-ER

– Lycopersicon esculentum Lupinus angustifolius – Alstroemeria spp. – Lycopersicon esculentum Lycopersicon esculentum – – – Capsicum annuum Musa spp. Tagetes erecta Pelargonium spp. Ocimum sanctum Tegetes erecta Lilium spp. – Lycopersicon esculentum Lycopersicon esculentum Amaranthus tricolor – Chrysanthemum morifolium Vigna unguiculata

UK Japan Australia Japan The Netherlands Hungary Japan China USA Australia France Australia China China India India India India USA Hungary Italy India Taiwan India USA

CMV subgroup II II II II II II II II II II II II II II II II II II IA IA IB IB IB OG OG

RNA 3 nt 96 96 96 96 96 96 95 95 96 94 95 95 95 95 – – – – 62 63 63 61 63 40 40

% identity at CP MP nt aa nt aa 95 94 96 96 95 95 94 96 95 95 96 95 94 95 95 95 94 93 68 68 70 69 70 41 42

94 93 93 93 91 91 92 94 93 94 93 94 93 92 93 93 91 90 79 79 78 76 78 42 46

98 98 98 98 98 98 97 97 98 96 97 97 97 97 – – – – 70 71 71 74 71 58 60

95 94 94 93 94 94 94 94 94 94 94 94 94 94 – – – – 82 79 81 78 81 63 63

Abbreviations used: CMV = Cucumber mosaic virus, OG = Outgroup, TAV = Tomato aspermy virus, PSV = Peanut stunt virus and – = information not available. J.Plant Dis.Protect. 5/2009


CMV-Car CMV-ALS CMV-Q CMV-241 CMV-Xb CMV-TE CMV-LY CMV-Tsh CMV-MT CMV-LS CMV-R CMV-Kin CMV-TN CMV-M2 CMV-Trk7

1 MDKSGSPNAS -------------------------------------------------------------------------------------------------------------------------------

11 RTSRRRRPRR ---------------------------------------------------------------Block A ----------------------------------------------------------------

21 GSRSASGADA -------------------------------------------------------------------------------------------------------------------------------

31 GMRALTQQML -L--------L--------L--------L--------L--------L--------L--------L---S----L--------L--------L--------L--------L--------L--------

41 RLNKTLAIDR --------G--------G--------G--------G--------G--------G--------GK--R----GK--R----GK--R----GK-------GK-------GK-------GK-------G-


76 KPPGIEKGSY ---E--------E--------E--------E--------E--------E--------E--------E--------E--------E--------E--------E--------E--------E------

86 FGRRLSLPDS -------------------------------------------------------------------------------------------------------------------------------

96 VTDYDKKLVS --------G-------------------------------------------------------------------------------------------------------------------Q--

106 116 RIQIRINPLP KFDSTVWVTV ---------- ------------------- ------------------- ------------------- -------A----------- ------------------- ---------Block B ---------- ------------------- ------------------- ------------------- ------------------- ------------------- ------------------- ------------------- ----------

126 RKVPSSSDLS --G----------------------------------------------------------------------------------------------------------------------------

136 VAAISAMFGD -------------------------------------------------------------------------------------------------------------------------T-----


151 161 LVYQYAASGV QANNKLLYDL ---------- K-T---------------- ------------------- ------------------- ------------------- ------------------- ------------------Block---------B ---------- ------------------- ------------------- ------------------- ------------------- ------------------- ------------------- --------N-

171 SEMRAEIGDM -----D--------D--------D---T----D---T----D--------D--------D--------D--------D--------D--------D--------D--------D--------D----

181 SKYTVLVKQK R--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YSR--A---YS-

201 FHVDVEHQRI L--------L--------L--------L--------L--------L--------L--------L--------L--------L--------L--------L--------L--------L---------

211 218 PISRMLPT ---------------------------------------------------------------------------------------------------

191 DDNLEKDEMV --K-----I--K-----I--K-----I--K-----I--K-----I--K-----I--K-----I--K-----I--K-----I--------I--------I--K-----I--K-----I--K-----I-

51 PTLNHPTFVG -----------------------------------------------------------------------------------------------------------------------------A-

61 SESCAPGYPF ----K---T----K---T----K---T----K---T----K---TR---K---T----K---T----K---T----K---T----K---T----K---T----K---T----K---T----K---T-

197

71 TSITL ---------------------------------------------------------

146 GNSPV ----G -------------------------------------------------K---

Fig. 4: Multiple alignments of CP amino acid residues of CMV-carrot (under study) with the selected strains of CMV subgroup II showing unique substitutions and conserved regions (shown in block A and B). CMV-carrot isolate has been abbreviated as CMV-Car and other strains are abbreviated as in Table 1.

4 Discussion Since carrot is an important consumable food crop grown commercially for its high nutritional value, identification of the virus infecting carrot became essential so that effective control/quarantine measures may be developed to minimize the losses and spread of the virus/es. Successful transmission of the virus on a number of test species by sap inoculations, positive transmission through A. gossypii in non-persistent and induction of systemic mosaic on C. sativus (a diagnostic host of CMV, FRANCKI et al. 1979) indicated the association of CMV with chlorotic mottle disease of carrot. Therefore, a set of J.Plant Dis.Protect. 5/2009

primers for CMV RNA3 was employed during RT-PCR, which resulted in an expected size of ∼2.2 kb amplicons, confirming the association of CMV. The analysis of sequence data obtained after cloning and sequencing of ∼2.2 kb amplicons revealed the presence of 2203 nucleotides of complete RNA3 sequence of CMV which included the CP and MP ORFs. Multiple sequence alignment of complete RNA3, CP and MP ORFs revealed high nucleotide and amino acid homology with CMV strains of subgroup II. Phylogenetic analysis also revealed its close relationship with CMV strains of subgroup II. The highest identities (> 96%) and close phylogenetic relationships with subgroup II clearly

198


A

64 AJ304399: CMV-ALS 16 EF202597: CMV-Tsh 2 AB189917: CMV-MT AJ585519: CMV-241 7 M21464: CMV-Q 34 98 AF127976: CMV-LS AB006813: CMV-M2 7 L15336: CMV-Trk7 12 11 AB176847: CMV-T 36 14 Z12818: CMV-Kin 97 Y18138: CMV-R 100

Subgroup II

AF198103: CMV-LY EU642567: CMV-Carrot

AF268598: CMV-Xb 98 EU665002: CMV-TE 42 AM114273: CMV-Le02 100 U66094: CMV-Sny 99 U20668: CMV-Fny 100 AF103991: CMV-Pepo

100

AJ831578: CMV-LI EF153733: CMV-Chry 100 EF593023: CMV-Amar U20219: CMV-Ix

100 77

98 100

Subgroup IA

Subgroup IB

D28780: CMV-Nt9 EF153734: CMV-Ts Y16926: CMV-Tfn NC_002040: PSV-ER EU163411: TAV-Luc

Out group

0.05

B

47

AJ304399: CMV-ALS AJ866272: CMV-Ger* AJ585519: CMV-strain-241

15

AF198103: CMV-LY AF268598: CMV-Xb

67 EU665002: CMV-TE 13 M21464: CMV-Q EF202597: CMV Tsh 73

EU600216: CMV-Oci* AB176847: CMV-TN

32

Subgroup II

L15336: CMV-Trk7 AB006813: CMV-M2

42 63 19

Z12818: CMV-Kin Y18138: CMV R

AJ585086: CMV-Lil* 37 AB189917: CMV-MT 99 29 29 AF127976: CMV-LS EU600214: CMV-carrot* 99

AM396983: CMV-Teg* 69 U20668: CMV-Fny AM114273: CMV-Le02 99 Y16926: CMV-Tfn 79 D28780: CMV-Nt9 EU163411: TAV-Luc NC_002040: PSV-ER

0.1

Subgroup IA Subgroup IB Out group

Fig. 5: Phylogenetic tree of CMVcarrot generated by MEGA 4 tool employing complete RNA3 sequence (EU642567) (a) and aa of coat protein (b) with the respective sequences of selected CMV strains of subgroup IA, IB and II. The virus isolate showed close phylogenetic relationship with CMV strains of subgroup II members reported worldwide. Indian strains included for study are marked by *. J.Plant Dis.Protect. 5/2009

Afreen et al.: Molecular identification of a Cucumber mosaic virus subgroup II isolate from carrot identified the virus as CMV of subgroup II. Although sequence data of CP gene is required for identification of the virus isolate into proper taxonomic position (SRIVASTAVA and RAJ 2004; VERMA et al. 2005a), complete RNA3 data is considered as important for better classification of various CMV strains (ROOSSINCK et al. 1999; DEYONG et al. 2005); therefore, the carrot isolate was studied at complete RNA3 level. Alignment of CP amino acid residues of CMV-carrot showed 13 unique substitutions which may have some impact on the coat protein orientation, symptoms expression, transmission as described earlier (PERRY et al. 1998). High similarities and conserved regions were observed among the CMV subgroup II members at CP level; therefore, this region may be utilized for specific detection of members of CMV subgroup II and for developing pathogen-derive resistance (PDR) in susceptible plants. It has been reviewed that CMV has a very broad host range among the plant viruses (PALUKAITIS and GARCIA-ARENAL 2003), but no reports of natural infection of CMV on carrot exist in literature. It is evidenced that strains of CMV subgroup IB occur dominantly in India (SRIVASTAVA and RAJ 2004), the report of CMV strain of subgroup IA infecting gladiolus (DUBEY et al. 2008), and subgroup II infecting Ornithogalum (VERMA et al. 2005a), Alstroemeria hybrids (VERMA et al. 2005b), Pelargonium (VERMA et al. 2006), Lycopersicon esculentum (SUDHAKAR et al. 2006) and Lilium spp. (MAHINGHARA et al. 2006) also exist in literature from India. In the present manuscript, we report the natural occurrence of CMV strain of subgroup II infecting carrot for the first time. The emergence of CMV subgroup II isolate in India may be the threat to cultivation of other agricultural crops. There are five reports on biological, serological and RT-PCR based identification of CMV isolates of subgroup II from India (VERMA et al. 2005a; 2005b; 2006; MAHINGHARA et al. 2006; SUDHAKAR et al. 2006) and nucleotide sequence data of coat protein genes of these isolates are available in GenBank database (AJ866272, EU600216, AJ585086, AM396983 and AJ745092). However, complete RNA3 genome based molecular characterization of any CMV isolate of subgroup II has not been done so far in India. Therefore, sequence information on complete RNA3 genome of CMV carrot isolate (EU642567) is the first report which would be an addition to the reports on complete RNA3 genome in other parts of the world.

Acknowledgements The authors are thankful to the chairman, Department of Botany, Aligarh Muslim University, Aligarh for providing the facilities under FIST funded project and to the director, National Botanical Research Institute, Lucknow for sequence analysis facility.

References DEYONG, Z., P. WILLINGMANN, C. HEINZE, G. ADAM, M. PFUNDER, B. FREY, J.E. FREY, 2005: Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format. J. Virol. Meth. 123, 101-108. DUBEY, V.K., AMINUDDIN, V.P. SINGH, 2008: First report of a subgroup IA Cucumber mosaic virus isolate from gladiolus in India. Aust. Plant Dis. Notes 3, 35-37.

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