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

Phytoparasitica (2013) 41:545–555 DOI 10.1007/s12600-013-0315-z

Molecular characterization of a Cucumber mosaic virus isolate associated with mosaic disease of banana in India Radha Vishnoi & Susheel Kumar & Shri Krishna Raj

Received: 20 March 2012 / Accepted: 31 May 2013 / Published online: 19 June 2013 # Springer Science+Business Media Dordrecht 2013

Abstract Severe mosaic accompanied by leaf and fruit deformation symptoms was observed on banana plants growing in three banana farms of Uttar Pradesh, India. The disease incidence was approximately 18– 25% at these locations during the three successive years from 2005 to 2007. The occurrence of Cucumber mosaic virus (CMV) was initially detected by bioassay, electron microscopic observations, Western blot immunoassay and RT-PCR. For molecular identification of virus, the RNA 1a, RNA 2b and RNA 3 genomic fragments were amplified by RT-PCR and sequenced. The sequence analysis of these genomic fragments revealed its highest identities and close relationships with Indian strains of CMVof subgroup IB; therefore, virus associated with the mosaic disease of banana was identified as an isolate of CMVof subgroup IB. In the limited reports existing from India, which provided preliminary serological or only coat protein-based identification of CMV infecting banana but the comprehensive studies were lacking. In the present communication, we present a detailed biological, serological and molecular characterization of CMVBanana for the first time from India. Keywords Bioassay . Cucumovirus . Electron microscopy . RNA3 genome . Sequence analysis . Serological detection . Subgroup IB R. Vishnoi : S. Kumar : S. K. Raj (*) Plant Molecular Virology Laboratory, CSIR-National Botanical Research Institute, Lucknow 226 006 U.P., India e-mail: [email protected]

Introduction Banana (Musa paradisiaca L.) is a dietary ingredient worldwide and has long been a staple food in India. The major cultivating states in India are Maharashtra, Uttar Pradesh (U. P.), Andhra Pradesh (A. P.), Karnataka and Tamil Nadu, with an annual total of ~23.2 million tons banana production (FAO 2008). Therefore, banana cultivation has high socio-economic and agricultural impact in India. Unfortunately, phytopathogens are the major setback to banana cultivation, among which Banana bunchy top virus and Cucumber mosaic virus (CMV) causing banana bunchy top and banana mosaic disease, respectively, are the major threats, jeopardizing the quality as well as quantity of fruits (Lockhart & Jones 2000). During our extensive surveys of the cultivating farms in northern India (Uttar Pradesh), banana plants exhibiting severe mosaic, leaf and fruit deformations accompanied by stunted plant symptoms were recorded. In India, banana mosaic disease was first described in 1980 by Mali & Rajegore on the basis of serology. Later, association of CMV with the banana mosaic disease was reported by Srivastava et al. (1995) based on immuno/nucleic acid probe assays. More recently, Khan and co-workers (2011) reported coat protein gene-based characterization of three isolates of CMV. However, comprehensive studies are still lacking. In this regard, the present communication will provide more insight into the detailed characterization of CMV infecting banana in India.

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Materials and methods Collection and maintenance of virus-infected banana plants Extensive surveys were conducted in three banana cultivating areas (Etawah, Kanpur and Lucknow) of Uttar Pradesh, India, to account the occurrence of banana mosaic disease during the years 2005–2007. Naturally infected banana plantlets exhibiting disease symptoms were collected and maintained in an insect-proof glasshouse at NBRI, Lucknow, for further study. Mechanical inoculations The mechanical inoculations were carried out on various host species, viz., Amaranthus tricolor, Chenopodium amaranticolor, C. album, Cucumis sativus, Datura metal, Lycopersicon esculentum, Nicotiana tabacum cv. ‘White Burley’ [all N. tabacum plants mentioned are of cv. White Burley], N. rustica, N. glutinosa, N. benthamiana and Petunia hybrida. The leaf tissue of a symptomatic banana plant was macerated in inoculation buffer in a ratio of 1:10 (w/v) as described by Noordam (1973). The homogenate obtained after squeezing through double-layered muslin cloth was rubbed onto carborandum pre-dusted leaves of recipient hosts (five of each species). The inoculated plants were observed for 30 days to record the local and systemic symptoms. Aphid transmission Aphid transmission was also attempted using Aphis gossypii Glover following the protocol described by Dheepa & Paranjothi (2010). Briefly, aphids were starved for 2 h, allowed acquisition access to a symptomatic leaf of banana for 2–3 min and then moved to five healthy banana plantlets and N. tabacum seedlings (~10 aphids/plant) for inoculation access of 2 h, before being sacrificed with 0.1% Confidor insecticide (Bayer Crop Science Ltd., India). The inoculated plants were then kept in an insect-proof glasshouse and observed for 6 weeks for symptom development, if any. Virus purification, electron microscopy and Western blot immunoassay The virus was purified by the procedure of A. Srivastava (2003, thesis, Lucknow Univ., India) using experimentally inoculated N. tabacum plants showing systemic mosaic symptoms. Then 250 g of infected leaves were finely ground in liquid nitrogen and used for purification. The virus pellet thus obtained was finally suspended in 200 μl storage

Phytoparasitica (2013) 41:545–555

buffer (5 mM sodium borate, pH 9.0, containing 0.5 mM EDTA) and kept at 4°C until further use. For transmission electron microscopy (TEM), copper grids (400 mesh, Canemco & Marivac Inc., Quebec, Canada) were coated with 50 μl of purified virus preparation for 2 min and washed with 10 mM phosphate buffer (pH 7.0) following three washings by sterile water. Virus particles were negatively stained with 2% uranyl acetate (pH 4.2) and excess stain was soaked away with a piece of clean filter paper and grids were allowed to air dry. Grids were observed in a TEM (Philips model 420, USA) at 80,000–100,000 magnification and photographed. For Western blot immunoassay, SDS-PAGE was performed using the leaf sap of naturally infected, experimentally inoculated banana and CMV-Gladiolus strain (Raj et al. 2002) as positive control. After SDSPAGE, the proteins were transferred onto a nitrocellulose membrane and processed with primary antibody (PVAS 242a, ATCC, USA), as described earlier by Renart & Sandoval (1984). The membrane was washed and blocked in blocking buffer TTBS (Tris 50 mM, NaCl 150 mM, Triton X-100, 0.1% and Tween-20, 0.05%) containing 5% non-fat, dry milk powder for 2 h at room temperature and then transferred to fresh blocking buffer containing 1:1000 diluted primary antibody (PVAS 242a, ATCC, USA) for 3 h. After three subsequent washing steps with TTBS, the blot was transferred to the buffer containing anti-rabbit IgG alkaline phosphatase conjugate (1:10,000 dilutions) and incubated at 4°C overnight. Finally, the protein bands were elucidated by color-development reaction on adding BCIP/NBT (Sigma, St. Louis, MO, USA) in the dark. The reaction was terminated by adding sterile water and the blot was dried. RNA isolation and RT-PCR of viral genes Total RNA from leaf tissue of infected and healthy banana was extracted following the procedure of S. Kumar (2009, thesis, Lucknow Univ., India). The total RNA isolated from 1 g of leaf tissue was dissolved in 50 μl sterile water and used for RT-PCR using CMV-CP primers (Acc. AM180922/AM180923). For molecular identification, the following primer pairs capable of amplifying RNA 1a (Forward: CAA GAG CGT ACG GTT CAA CCC CTG CCT, Reverse: TCA AAA CAC CCT (CT)CC GCC CAC TCG TT), RNA 2b (Forward: ATG GAA TTG AAC GCC GGA GGC GCA ATG, Reverse: TCA AAA CAC CCT (CT)CC GCC CAC TCG TT) and RNA 3 (Forward: GAT CCC CGG GTA


ATC TTA CCA CTG TG, Reverse: TGG TCT CCT TTT GGA GGC CCC ACG A) were used. cDNA synthesis was performed separately for each fragment in a 20 μl reaction mixture containing 1 μg of RNA template, 1 μl reverse transcription buffer, 1 mM each dNTPs, 25 pM reverse primer (gene specific), 20 U RNA guard (MBI Fermentas, Hanover, MD, USA) and 200 U of Revert Aid H-minus MMuLV Reverse transcriptase (MBI Fermentas) following the manufacturer’s instructions. Thereafter, PCR was carried out separately in a 50 μl reaction mixture containing: 10 ng cDNA as template, 1 μl Pfu DNA polymerase buffer, 200 μM dNTPs, 25 pM forward and reverse primers and 3 U Pfu DNA polymerase (MBI Fermentas) in Peltier thermal cycler PTC200 engine (MJ Research, Waltham, MA, USA) to amplify the respective fragments. The PCR conditions were: 94°C for 3 min (1 cycle); 94°C for 1 min, 56°C for 1 min, 72°C for 90 sec for RNA 1a and RNA 3 and 20 sec for RNA 2b (each 30 cycles); final extension for 5 min at 72°C. Amplicons were analyzed on 1% agarose gel with DNA marker (λ DNA EcoRI/HindIII marker, Genei Pvt. Ltd, Bangalore, India). Cloning and sequencing The A-tail was added to the PCR products using Klenow polymerase (Genei Pvt. Ltd., Bangalore, India). The products were purified using QIAquick Gel Extraction kit (Qiagen GmbH, Hilden, Germany) and ligated into pGEM-T Easy vector system-I (Promega Corporation, Fitchburg, WI, USA). The competent Escherichia coli DH5α cells were transformed and clones were screened on Luria agar plates supplemented with 100 mg l-1 Ampicillin, 80 μg ml-1 X-gal and 0.5 mM IPTG. White colonies were screened and three such positive clones of each fragment were sequenced. Sequence data obtained were analyzed, consensus sequence of three clones of each gene was determined and deposited in the GenBank database. Computational analysis of sequence data The data were analyzed by BLASTn (http://www.ncbi.nlm.nih. gov/BLAST) and then compared with various existing sequences of CMV strains available in the GenBank database. To obtain the sequence identities, matrix for pair-wise alignment of selected CMV strains was done using the Genomatix DiAlign 2.1 program (http://www.genomatix.de/cgi-bin/dialign/dialign.pl). The open reading frame (ORF) in sequenced data

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was predicted by ORF finder program (http://www. ncbi.nlm.nih.gov/projects/gorf/) to find in frame AUG-start and TAG-termination codons and translated into putative amino acids using the ExPasy tool (http:// www.expasy.org/tools/dna.html). Phylogenetic analyses were done employing CMV strains of subgroups IA, IB and II using the Molecular Evolutionary Genetics Analysis tool (MEGA v4.2) with 1000 replicates bootstrapping. Phylograms were generated using the Neighbor Joining (NJ) method and viewed by the NJ plot program. Peanut stunt virus (PSV, AY775057) and Tomato aspermy virus (TAV, EU163411) were considered as referenced out-group members of the genus Cucumovirus for rooting of the dendrogram.

Results Natural symptoms and disease incidence Banana (Musa sp.) is widely grown in India and U. P. is one of the major producers of quality bananas. During surveys of banana cultivation fields of U. P. in three successive years (2005–2007) the natural occurrence of mosaic accompanied by fruit deformation symptoms (Fig. 1) was observed with about 18–25%, 20–22% and 19–24% disease incidences at Etawah, Kanpur and Lucknow, respectively, in U. P. Mechanical inoculations The causal virus was successfully transmitted by mechanical inoculations to a number of test species. The necrotic lesions were evoked on C. amaranticolor (4/5) (Fig. 2a), C. album (3/5) and necrotic rings on N. tabacum (5/5) (Fig. 2b) and N. rustica (5/5) (Fig. 2c). The virus induced systemic blisters on N. rustica (5/5) (Fig. 2d) and N. tabacum (5/5) (Fig. 2e), shoestring on N. glutinosa (4/5) (Fig. 2f) and L. esculentum (3/5) (Fig. 2g), and systemic mosaic on C. sativus (5/5) (Fig. 2h). Neither local nor systemic symptoms were obtained on A. tricolor, D. metal, N. benthamiana or P. hybrida even 30 days post-inoculation. Aphid transmission During aphid transmission, banana virus was successfully transmitted from the naturally infected banana to healthy banana and N. tabacum plants through A. gossypii vectors in a non-persistent manner. The transmission rate was 40% (2/5) in banana and 60% (3/5) in N. tabacum plants. The inoculated banana produced mosaic symptoms similar to naturally infected plants.

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Fig. 1 Severe mosaic symptoms (a) observed in naturally infected banana plants and a close view of infected leaf (b)

Electron microscopy and Western blot immunoassay The purified virus preparation obtained after following the protocol described by A. Srivastava (2003, thesis, Lucknow Univ., India) and used for EM revealed isometric cored virus particles of ~28 nm (in diameter) (Fig. 3). Western blot immunoassay with CMV antibody (PVAS 242a) using leaf sap of naturally infected as well as experimentally inoculated banana plants showed positive signals of antibody-antiserum reaction (Fig. 4, lanes 2, 3). The 26 kDa band closely matched the molecular mass of coat protein of CMVGlad (Raj et al. 2002) taken as positive control (Fig. 4, lane 1). These results verified the presence of CMV coat protein of 26 kDa in infected banana. RT-PCR based detection of virus from banana RTPCR using the total RNA from infected banana samples and CMV-CP gene-specific primers resulted in the expected amplicons of ~650 bp in the samples collected from three locations: Etawah, Kanpur and Lucknow, but not in an asymptomatic healthy sample. The positive amplification for expected size bands indicated the infection of CMV in banana samples collected from three locations of U. P. Cloning and sequencing of genomic fragments for molecular characterization Since molecular characterization is essential for authentic identification of the virus isolate, RNA 1, RNA 3 and RNA 2b genomic fragments of banana virus isolated from Lucknow were amplified by RT-PCR. The total RNA preparations isolated from symptomatic and asymptomatic banana leaves were subjected to cDNA synthesis using 1 μg RNA with 100 pM reverse primer for

RNA 1, RNA 3 and RNA 2b genes. PCR with genespecific forward and reverse primers resulted in the expected size amplification of ~3.3 Kb, 2.2 Kb and 350 bp bands, respectively. These amplicons were cloned in pGEM-T Easy vector and positive clones for these fragments were sequenced and the data obtained from three clones of each were assembled, and analyzed in their entirety to resolve any ambiguity to obtain the consensus sequence. Genomic organization of Lucknow isolate of banana virus The analysis of sequence data of RNA 1 of banana virus isolate revealed 3358 nucleotides which contained 2982 nucleotides of RNA polymerase subunit Ia gene translating into 993 amino acid residues. The sequences were deposited in GenBank under the accession EU159528. Analysis of sequence data of RNA 2b gene revealed the presence of 336 nucleotides encoding 112 amino acid residues (Acc. EU417842). RNA 3 genome was found to be 2219 nucleotides during sequence analysis and consisted of two ORFs: MP gene of 840 nucleotides putatively translating 229 amino acids and CP gene of 657 nucleotides translating 218 amino acid residues. Both the ORFs were separated by a 300 nucleotide-long intergenic region and flanked by 5’un-translated region (UTR) and 3’UTR of 122 and 302 nucleotides, respectively. The sequence data of RNA 3 were deposited in the GenBank under the accession EF178298. Sequence analysis of RNA 1 During BLASTn analysis, RNA 1 (EU159528) showed the highest 93% sequence identity with CMV-CLW2 strain of cucumber (JN054636) from Malaysia, 92% with CMV- IA


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Fig. 2 Symptoms evoked by the inoculation of banana virus isolate on various host plants. The virus induced necrotic local lesions on Chenopodium amaranticolor (a) and necrotic rings on Nicotiana tabacum cv. White Burley (b) and N. rustica (c);

systemic blistering on N. rustica (d) and N. tabacum cv. White Burley (e); shoestring on N. glutinosa (f) and L. esculentum (g); and systemic mosaic on Cucumis sativus (h)

(AB042292) from Indonesia, and 90% with CMV strains CTL (EF213023) and Phy (DQ402477) from China. Sequence alignment of RNA 1 gene with available CMV strains by Genomatix DiAlign revealed highest 89% nucleotide and 95% amino acid identity with the CMV strain from Indonesia (AB042292, subgroup IB), whereas tomato strain (GU111227) from New Delhi, India, showed lower 85% nucleotide and 90% amino acid sequence identities. During alignment of RNA 1a, sequence variability in its genome was revealed, as expected with some unique amino acid substitutions.

Sequence analysis of RNA 2b During BLASTn analysis, the RNA 2b gene (EU417842) of the virus isolate showed highest 99% sequence identity with CMV isolate of Solanum melongena (HM143913) and 97% with CMV isolates from Chrysanthemum morifolium (EU450890) and petunia (JF798577) from India. The RNA 2b gene showed highest 94% nucleotide and 92% amino acid identities with the IARI strain of CMV (EU006067). However, it showed only 82–87% sequence identities with other strains of subgroup IB.

Fig. 3 Electron micrograph of a leaf dip preparation of naturally infected banana showing isometric cored virus particles (indicated by black arrow heads). Bar = 60 nm

Sequence analysis of RNA 3 During BLASTn analysis, the RNA 3 of Lucknow isolate (EF178298) showed highest 96% sequence identity with CMV-CLW2 isolate of cucumber from Malaysia (JN054635) and Indian CMV isolates from Rauwolfia (CMV-R, EF593025),

Fig. 4 Western blot immunoassay using CMV antiserum (PVAS 242a) showing 26 kDa viral protein in the leaf sap of naturally infected banana. Lanes M: protein marker, 1: CMVGlad as a positive control (Raj et al. 2002), 2: naturally infected banana, and 3: experimentally inoculated banana

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Petunia (JN642676), Jatropha (EF593026), Amaranthus (CMV-A, EF593023), Datura (CMV-D, EF593024), and 95% with CMV strain of Musa sapientum from Japan (AB046951). The RNA 3 was also aligned to observe the sequence identities in their entirety, which revealed highest 94% nucleotide identities with Indian isolates of CMV-A, -D and -R strains (Table 1); however, lower 88–93% identities were recorded with other CMV strains of subgroup IB.

The ORFs and non-translating regions in RNA 3 were also aligned separately to explore the deeper knowledge of sequence identity. During amino acid alignment of MP, it shared 95–100% amino acid identities with the strains of subgroups IA and IB. Similarly, 5’UTR, 3’UTR and inter domain region (IR) of banana virus isolate shared, respectively, 85–99%, 83–100% and 91–100% nucleotide identities with subgroup IB strains of CMV. The IR and

Table 1 Sequence identity of CMV-banana isolate (EF178298) obtained after the multiple alignments with the selected strains of CMV subgroup IA, IB and II based on Genomatix DiAlign analysis GenBank Accession

Abbreviation Natural host

Country/ Location

Sub % nt identity obtained at the level of group RNA 3 5’UTR MP IR CP nt

nt

nt

3’UTR

nt

aa

nt

aa nt

97

100 99 99 100

EF593023

CMV-A

Amaranthus tricolor

India

IB

94

99

98

EF593024

CMV-D

Datura innoxia

India

IB

94

99

100 100 100 98 95 100

EF593025

CMV-R

Rauwolfia serpentina

India

IB

94

99

99

97

100 98 94 100

EF593026

CMV-J

Jatropha curcas

India

IB

93

99

99

98

100 98 95 100

EF153733

CMV-Ch

Chrysanthemum morifolium India

IB

93

96

95

97

94

95 98 90

EF216865

CMV-YN

Brassica chinensis

China

IB

93

97

93

96

94

89 93 97

EF216867

CMV-Cb-7

Lycopersicon esculentum

China

IB

93

97

93

96

94

89 93 98

Y16926

CMV-Tfn


USA

IB

93

97

92

95

90

90 93 96

AB042294

CMV-IA

─

USA

IB

92

93

94

96

93

90 94 95 89 93 97

DQ412732

CMV-Phy

─

China

IB

92

97

93

96

95

D28780

CMV-Nt9

─

Taiwan

IB

92

97

92

95

91

90 93 97

EF153734

CMV-Ts


India

IB

92

97

92

95

90

90 99 97

AM183116

CMV-P1-1


Barcelona

IB

91

95

92

95

90

89 92 94

AF268597

CMV-PE

Passion flower

China

IB

91

95

93

95

93

89 92 96

EF213025

CMV-CTL

Brassica chinensis

China

IB

93

98

93

96

91

91 94 91

U20219

CMV-Ix

Ixora sp.

Philippines

IB

89

92

84

93

85

90 91 90

AB369272

CMV-P

Pepper sp.

South Korea IB

88

85

91

96

82

88 92 90

AJ511990

CMV-Ns

Nicotiana glutinosa

Hungary

IB

88

85

91

96

83

89 93 91

D10538

CMV-Fny

Cucurbita pepo

USA

IA

87

85

91

97

82

89 93 90

AB369270

CMV-V

─

South Korea IA

87

85

91

96

82

88 93 90

AM114273

CMV-LeO2


Hungary

IA

87

85

91

96

82

88 93 90

AJ276481

CMV-Mf

─

South Korea IA

88

84

91

96

83

90 93 90

Y18137

CMV-I17F

─

France

IA

88

85

91

96

82

89 92 90

U66094

CMV-Sny

Cucurbita pepo

USA

IA

87

87

91

96

82

89 92 92 86 91 80

AJ831578

CMV-LI

Lilium longiflorum

India

IA

87

54

72

96

61

M21464

CMV-Q

─

Australia

II

63

54

75

76

59

87 90 59

Z12818

CMV-Kin

─

Scotland

II

62

54

73

81

61

71 79 60

AF227976

CMV-LS

NC_002040 ER-PSV

─

USA

II

62

53

75

82

66

71 78 59

Vigna unguiculata

USA

OG

37

33

55

63

37

43 44 55

Abbreviations used: CMV= Cucumber mosaic virus, OG= Out group, PSV= Peanut stunt virus, ─ not available


3’UTR were found to be more conserved compared with 5’UTR. BLASTn analysis of the CP gene of banana virus under study (DQ028777) showed highest 99% sequence identity with Indian strains of CMV-A from Amaranthus (EF593023) and CMV-Ts from tomato (DQ141675). Our isolate also showed variable sequence identity with three CMV banana strains: 93% with the Karnataka isolate (AM055602), 98% with the Maharashtra (DQ640743) and 95% with the U. P. isolate (AM158321). Multiple alignments of amino acid sequences of CP of CMV banana isolate under study with the selected members showed highest 99% sequence identity with Indian strains of CMV-A (EF593023) and 98% with CMV-Ch (EF153733). Interestingly, banana isolate has unique substitutions at three positions: R/K76, S/T120 and M/V187 (Fig. 5). These unique substitutions were also found in two Indian isolates of CMV (CMV-A and -Dat) reported earlier (Srivastava & Raj 2004). Besides this, CP gene showed highest 99% nucleotide and amino acid identities with CMV-A and -Ts strains, whereas 98% nucleotide and 95%

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amino acid sequence identities with CMV-Ch, -D, -J and -R. As expected, identities were decreased to 91– 97% at nucleotide level and 90–93% at amino acid level with CMV-subgroup IA members (Table 1). Phylogenetic relationship of Banana virus isolate In order to know the phylogenetic and evolutionary relationships of the banana virus isolate with the established CMV strains of subgroups IA, IB and II, analysis was carried out using MEGA tool employing CP, RNA 3, 2b and 1a sequences. During phylogenetic analysis of RNA 3 (EF178298), the virus isolate under study clustered within subgroup IB and showed close relationships with Indian isolates of CMV-A (EF503023), -D (EF593024), -J (EF593026), -R (EF593025) and -Ch (EF153733). However, subgroups IA and II strains showed distant relationships with the banana isolate (Fig. 6). In phylogenetic analysis of deduced amino acid residues of coat protein, banana virus isolate clustered with CMV strains of subgroup IB and exhibited the closest relationship with the CMV-Ch (DQ028777) strain of subgroup IB (Fig. 7). However, it clustered slightly distantly with

Fig. 5 Coat protein-based multiple sequence alignment of banana virus isolate with the selected members of CMV of subgroups IA, IB and II strains reported. - Identical nucleotide sequences have been shown by dashed line and differences by letters.

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the CMV banana strains reported from Maharashtra (DQ640743), Karnataka (AM055602) and Kerala (AY125575) in India. RNA 2b-based phylogenetic analysis of banana virus under study with the selected strains of CMV (subgroups IA, IB and II), revealed also closeness with the subgroup IB strains and clustered together with CMV-Ch (EU118810) and -IARI (EU006067) strains (Fig. 8). Based on CP, RNA 3 and 2b, all Indian strains of CMV clustered within subgroup IB and formed a separate clade, indicating that Indian strains are closely related to each other and grouped within subgroup IB. However, phylogenetic analysis of the RNA 1a genome with all three subgroups of CMV (IA, IB and


II) did not indicate a significant relationship with any of the subgroup isolates and failed to produce the subdivision among CMV subgroups.

Discussion During virus transmission tests, the causal virus was successfully transmitted by mechanical inoculations to a number of test species. The systemic mosaic on C. sativus was also obtained, indicating that the causal virus is an isolate of Cucumber mosaic virus (CMV), because C. sativus is a well known diagnostic host for CMV (Owen & Palukaitis 1988). Banana virus induced

Fig. 6 Phylogenetic analysis of banana virus isolate (highlighted in gray box) based on RNA 3 genomic fragment. The tree has been generated by MEGA v4.1 and the evolutionary history was inferred using the Neighbor-Joining method with 1000 replicate bootstraps.


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remarkable differences with variable symptoms as compared with CMV-Ch and -D. These variations in symptoms produced might be due to the natural variation/ mutation continuously occurring in virus populations, responsible for giving rise to new strains – as suggested earlier (Palukaitis & Garcia-Arenal 2003). CMV has been reported to be transmitted in nature by aphid vectors (Noordam 1973) and during this study, banana virus was also transmitted from the naturally infected banana to healthy banana and N. tabacum plants through A. gossypii vectors in a non-persistent manner, in accordance with a previous study (Aglave et al. 2007), where CMV causing mosaic and chlorosis of banana in Marthwada, India, was found to be

transmitted by A. gossypii and A. craccivora. The virus isolate from banana was successfully transmitted on banana with 40% transmission rate and inoculated banana plants produced mosaic symptoms similar to naturally infected plants, thereby fulfilling Koch’s postulates. During EM of virus preparations, isometric cored virus particles ~28 nm in diameter were observed – which is a characteristic feature of CMV as described by Francki et al. (1979) – and indicated that the virus is CMV. Western blot immunoassay showed positive signals with CMV antiserum (PVAS 242a, ATCC, USA) and the 26 kDa band was observed which closely matched the molecular mass of coat protein

Fig. 7 Phylogenetic analysis of coat protein amino acid residues of banana virus isolate (in bold) with various CMV strains reported worldwide on banana except a few on other hosts

(marked by *). The tree has been generated by MEGA v4.1 and the evolutionary history was inferred using the NeighborJoining method with 1000 replicate bootstraps.

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of CMV-Glad (Raj et al. 2002) taken as positive control, and verified the presence of CMV coat protein of 26 kDa in infected banana. Based on mechanical transmission on various host species, positive aphid transmission in a non-persistent manner, presence of isometric cored particles of 28 nm (in diam) during EM, positive reaction with antiserum to CMV and 26 KDa viral protein band during Western blot immunoassay, positive amplification of expected size (~650 bp) band during RT-PCR with CMV coat protein gene-specific primers and findings of the sequence data analysis – the virus isolate under study associated with mosaic disease of banana has been identified as a strain of CMV of subgroup IB which has close relationships with Indian CMV strains.


The three unique substitutions at positions R/K76, S/T120 and M/V187 obtained in the CP region of banana virus under study during amino acid alignments were also observed earlier in two Indian isolates of CMV (CMV-A and -Dat) belonging to subgroup IB (Srivastava & Raj 2004). Alterations at these positions might suggest some functional importance to provide selective biological advantages for survival of CMV strains and be regarded significant for virus evolution, as suggested earlier by Bonnet et al. (2005). During the phylogenetic analysis of amino acid sequences of the CP region of the isolate under study, along with all the CMV banana strains from India available in the GenBank database (Acc. AM158321 & DQ152254: Lucknow, UP; DQ640743: Maharashtra;

Fig. 8 RNA 2b based phylogenetic analysis of banana virus under study by MEGA v4.1. The evolutionary history was inferred using the Neighbor-Joining method with 1000 replicate bootstraps.


AM055602: Karnataka; AY125575: Kerala), clearly revealed that they belong to CMV subgroup IB; however, prior to this report there has been no invasion of other CMV subgroups (IA & II) in India. Recently, Chou and co-workers (2009) characterized ten Taiwanese isolates of CMV from banana based on CP sequence and reported four different pathotypes from all subgroups which showed the existence of subgroups I and II in Taiwan. There are records of serological detection (Mali & Rajegore 1980) or coat protein-based molecular characterization of CMV infecting banana from India (Srivastava et al. 1995). Recently in India, Khan et al. (2011) reported characterization of three CMV isolates from Karnataka, Maharashtra and Uttar Pradesh infecting banana based only on coat protein gene analysis and concluded that these CMV strains belong to subgroup IB (Khan et al. 2011). However, detailed sequence information of the viral genome of CMV infecting banana and its molecular characterization was lacking. The present study provides comprehensive sequence information on RNA 1a, RNA 3 and RNA 2b genomic fragments and detailed characterization of CMV associated with banana mosaic disease. Banana mosaic disease caused by CMV has become an important threat to the banana industry in Taiwan (Chou et al. 2009). The present study also suggests that banana mosaic disease may be a serious threat to banana cultivation in India in the near future. In addition, the disease can also have important indirect effects by restricting germplasm movement and by predisposing plants to damage by other biotic and abiotic stress factors. Acknowledgments The authors are thankful to the Director, National Botanical Research Institute, for the facilities to conduct this work and to the University Grant Commission (U.G.C.), New Delhi, for fellowships to Radha Vishnoi.

References Aglave, B. A., Krishnareddy, M., Patil, F. S., & Andhale, M. S. (2007). Molecular identification of a virus causing banana chlorosis disease from Marathwada region. International Journal of Biotechnology and Biochemistry, 3, 13–23.

555 Bonnet, J., Fraile, A., Sacristán, S., Malpica, J. M., & GarcíaArenal, F. (2005). Role of recombination in the evolution of natural populations of Cucumber mosaic virus, a tripartite RNA plant virus. Virology, 332, 359–368. Chou, C.-N., Chen, C.-E., Wu, M.-L., Su, H.-J., & Yeh, H.-H. (2009). Biological and molecular characterization of Taiwanese isolates of Cucumber mosaic virus associated with Banana mosaic disease. Journal of Phytopathology, 157, 85–93. Dheepa, R., & Paranjothi, S. (2010). Transmission of Cucumber mosaic virus (CMV) infecting banana by aphid and mechanical methods. Emirates Journal of Food and Agriculture, 22, 117–129. F. A. O. (2008). FAOSTAT: ProdSTAT: Crops. UN Food & Agriculture Organization. Francki, R. I. B., Mossop, D. W., & Hatta, T. (1979). Cucumber mosaic virus. CMI/AAB Descriptions of Plant Viruses, 2, 213. Khan, S., Jan, A. T., Aquil, B., & Haq, Q. M. R. (2011). Coat protein gene based characterization of Cucumber mosaic virus isolates infecting banana in India. Journal of Phytology, 3, 94–101. Lockhart, B. E. L., & Jones, D. R. (2000). Banana mosaic. In D. R. Jones (Ed.), Diseases of banana, abaca and enset (pp. 256–263). Wallingford, UK: CAB International. Mali, V. R., & Rajegore, S. B. (1980). A Cucumber mosaic virus disease of banana in India. Phytopathologie Zeitung, 98, 127–136. Noordam, D. (1973). Dilution end-point determination. In Identification of plant viruses: methods and experiments. Wageningen, the Netherlands: PUDOC, Center for Agricultural Publishing and Documentation, Owen, J., & Palukaitis, P. (1988). Characterization of Cucumber mosaic virus. I. Molecular heterogeneity mapping of RNA 3 in eight CMV strains. Virology, 69, 496–502. Palukaitis, P., & Garcia-Arenal, F. (2003). Cucumber mosaic virus. Descriptions of plant viruses. CMI/AAB, No. 400:1-23. Raj, S. K., Srivastava, A., Chandra, G., & Singh, B. P. (2002). Characterization of Cucumber mosaic virus isolate infecting Gladiolus cultivars and comparative evaluation of serological and molecular methods for sensitive diagnosis. Current Science, 83, 1132–1136. Renart, J., & Sandoval, I. V. (1984). Western blots. Methods in Enzymology, 104, 455–460. Srivastava, A., & Raj, S. K. (2004). High molecular similarity between Indian isolates of Cucumber mosaic virus suggests a common origin. Current Science, 87, 1126–1131. Srivastava, A., Raj, S. K., Haq, Q. M. R., Srivastava, K. M., Singh, B. P., & Sane, P. V. (1995). Association of a Cucumber mosaic virus strain with mosaic disease of banana, Musa paradisiaca – an evidence using immuno/nucleic acid probe. Indian Journal of Experimental Biology, 33, 986–988.