Badnavirus, Potyvirus and Cucumovirus) in Canna Species in India

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Research Article *Corresponding author SK Raj, Plant Molecular Virology Laboratory, Council of Scientific and Industrial Research (CSIR)-National Coexistence of Three Virus Botanical Research Institute (NBRI), Lucknow-226001, UP, India, Tel: 91-522-2297950; Fax: 91-522-2205839; Email:

Genera (Badnavirus, Potyvirus Submitted: 27 January 2016 Accepted: 18 April 2016 and Cucumovirus) in Canna Published: 20 April 2016 Copyright Species in India © 2016 Raj et al. OPEN ACCESS Aarti Kumari1, Rashmi Raj1, Susheel Kumar1, Puneet Singh Chauhan2 and SK Raj1* Keywords • Canna 1 Plant Molecular Virology Laboratory, Council of Scientific and Industrial Research • Canna yellow mottle virus (CSIR)-National Botanical Research Institute (NBRI), India • Bean yellow mosaic virus 2 Division of Plant Microbe Interactions, Council of Scientific and Industrial Research • Cucumber mosaic virus (CSIR)-National Botanical Research Institute (NBRI), India

Abstract Canna is an important ornamental plant recognized for attractive flowers and variegated foliages. Canna plants of ten economically important cultivars (Allegheny, Bengal Tiger, Black Knight, Eileen Gallo, Encaster, Golden Girl, Golden Yellow Lucifer, Kanchan, New Delhi Yellow, and Red President) of canna repository at CSIR-NBRI, India exhibiting mild to severe yellow mosaic mottle and vein streak symptoms were observed during surveys from January 2012 to January 2014. The disease incidences varied from 8.0-74.4%. Occurrence of single, double, and triple infections of Canna yellow mottle virus (CaYMV), Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV) were detected in these cultivars by PCR/RT-PCR using their respective degenerate primers. The coexistence of CaYMV, BYMV and CMV was confirmed by sequencing of their cloned PCR products. Further, a high genetic diversity was observed amongst them and with other strains of CaYMV, BYMV and CMV in phylogenetic analysis. The coexistence of CaYMV, BYMV, and CMV in canna is being reported for the first time from India.

ABBREVIATIONS Canna is reported to be susceptible to many phytopathogens such as: Xanthomonas cannae Cordana veriscolor CaYMV: Canna yellow mottle virus; BYMV: Bean yellow mosaic Puccinia thaliae Canna virus; CaYSV: Canna yellow streak virus; CMV: Cucumber Mosaic host of viruses worldwide. Canna [6], yellow mottle virus (CaYMV) [7] Virus; TAV: Tomato Aspermy Virus; PCR: Polymerase Chain is reported to infect [8]. Besides several this, canna species species and are is commonly known as Reaction; RT-PCR: Reverse Transcription-Polymerase Chain Cucumber mosaic Reaction, TEM: Transmission Electron Microscopy virus (CMV) and Tomato aspermy virus associated with leaf mottling symptoms [9-14]. INTRODUCTION Bean yellow mosaic virus (TAV) are knownCanna to mottle infect Cannas (Canna spp., family Cannaceae) are perennial plants viruscanna and induces mosaic and streaks symptoms [9,15-17].Canna yellow grown in tropical and sub-tropical countries including India. streak virus (CaYSV)(BYMV), has been previously reported inknown canna as causing yellow They are largely used in borders of beds for their colourful [13], is also reported to infect canna [9,18]. is common in canna, while co-infection of two different genera streak disease [19,20]. The infection of viruses of single genus foliages and flowers, besides being grown in lawns, parks, and other public places. Therefore, canna has significance due to their thehas infectionalso been of reported CaYMV, [9,21],CMV, and however, BYMV thein ten coexistence important of Canna three large shiny foliage and attractive flowers of red, yellow, orange, cultivarsviruses of maintaineddifferent genera in canna is not repository known in India. of CSIR-NBRI, Here we report India. pink colour or any combinations of these colours [1]. Canna rhizome is consumed as food being rich in starch [2,3] and is Three (Allegheny, Black Knight, and Eileen Gallo) cultivars, out also used for bio-ethanol production [4]. In recent years, cannas virus infection in canna may aid in their early detection, searching of ten, were co infected by CaYMV, CMV and BYMV. Knowledge of have got attention of breeders and floriculturists to develop new of virus-free propagating material or disease management. commercially important varieties [5].

Cite this article: Kumari A, Raj R, Kumar S, Chauhan PS, Raj SK (2016) Coexistence of Three Virus Genera (Badnavirus, Potyvirus and Cucumovirus) in Canna Species in India. Ann Virol Res 2(1): 1008. Raj et al. (2016) Email:

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Central Bringing Excellence in Open Access MATERIALS AND METHODS employing Neighbour-Joining (N-J) method and viewed by the replicates bootstrapping [26]. ThePineapple dendrogram bacilliform was erectifolius generated virus Sugarcane bacilliform virus Three consecutive surveys in January month of 2012, 2013, N-J plot program of MEGA tool. and 2014 were conducted for scoring the virus like diseases for rooting (PBCErV, of the EU377376)phylogenetic and tree. in ten different canna cultivars (Allegheny, Bengal Tiger, Black (SBCV, FJ439817) strains have been used as out group members Knight, Red President, Kanchan, Eillen Gallo, New Delhi° Yellow, RESULTS AND DISCUSSION Golden Girl, Encastero and Golden Yellow Lucifer) maintained at repositoryassessed by of visual CSIR-NBRI, observation Lucknow, of symptoms.India (latitude: The 26symptomatic 51’N and longitude: 80 55’E). Disease incidence in these ten cultivars was mild to severe symptoms were observed on leaves of Allegheny, and asymptomatic (control) leaf samples were collected and During surveys (January 2012-2014) of canna repository, used for virus detection. Bengal Tiger, Black Knight, Red President, Kanchan, Eileen Gallo, electron microscopic (TEM) study was conducted using leaf dip New Delhi Yellow, Golden Girl, Encaster and Golden Yellow For morphological virus identification, transmission Lucifer cultivars (Figure 1). Visual observation of symptoms Eileen in these cultivars revealed 8.0 to 74.4% disease incidences (Table preparations as described earlier [21]. The virus particles on grid 1). TEM analysis of infected Allegheny, Black Knight, and were negatively stained with 2% Uranyl acetate and observed in Gallo samples revealed the presence of typical bacilliform, a TEM (Philips EM420, USA) at 35-100 K magnification. andflexuous suggest rods their and co cored infection. spherical Bengal virions Tiger measuring cultivar revealed about canna leaf samples from ten cannas cultivars (three samples 130x30 nm, 600x11 nm, and 28 nm (in diameter), respectively fromFor each molecular cultivar) detection were subjected of viruses, for a total PCR/RT-PCR. of 30 symptomatic The total the mixed infection of bacilliform and flexuous rod shaped virus particles. The four cultivars: Encaster, Golden Girl, Golden Yellow DNA and RNA were isolated independently from leaf samples Lucifer and Red President showed the presence bacilliform virus using GenElute Plant Genomic DNA Miniprep kit (Sigma-Aldrich, particles and suggest badnavirus infection only, while Kanchan MO, USA) and RNeasy Plant Mini Kit (Sigma-Aldrich, MO, USA), particles only and suggest potyvirus infection. cultivar showed the presence of flexuous rod shaped virus wererespectively directly following subjected the for PCRmanufacturer’s with Badnavirus instructions degenerate and used as templates for PCR and RT-PCR. The DNA preparations Badnavirus degenerate primers In PCR/RT-PCR analyses, 22 samples showed expected size bpprimers band. The[22] RNA following preparations their were recommended reverse transcribed amplification into amplification of ~600 bp with conditions whichPotyvirus expected to directCucumovirus the amplification of ~600 from Allegheny, Bengal Tiger, Black Knight, Eileen Gallo, Encaster, bp)Golden with Girl, Potyvirus Golden degenerateYellow Lucifer primers and Red from President Allegheny, cultivars. Bengal cDNA with [23] and [24] downstream The 9 samples showed amplification of expected size (~1600 theirdegenerate instructions. primers Further, independently PCR were using conducted GoScript™ following Reverse the conditionsTranscription described System earlier (Promega for PotyvirusCorp., Madison, USA)Cucumovirus following withTiger, Cucumovirus Black Knight, Eileen Gallo, Encaster and Kanchan cultivars, whereas, 5 samples showed expected size ~900 bp amplicon [23] and been shown to display genus the specific obtained primers amplicons from Allegheny, from ten cannaBlack [24] detection, respectively in a SureCycler 8800 Thermal Cycler Knight and, Eileen Gallo cultivars. A representative figure has (Agilent Technologies, USA). The sizes of products were assessed Yellow cultivar failed to produce any band with any of these on 1% agarose gel with 1 kb DNA size marker (Thermo-Fisher cultivars (Figure 2a-c). All the three leaf samples of New Delhi Scientific Inc., Pittsburgh, USA). degenerate primers. Molecular analysis confirmed why no virus The expected size PCR products from the gel were eluted particles could be observed in New Delhi Yellow cultivar during using Wizard®® SV Gel and PCR Clean-Up System (Promega Corp., -T Easy Vector System-I (Promega Corporation, Madison, TEM. Three cultivars: Allegheny, Black Knight and Eileen Gallo Madison, USA). The purified products were cloned separately into degenerate primers and suggest triple infection in them. Bengal Escherichia coli produced expected size amplicons with all three genus specific pGEM Tiger cultivar also showed the amplicons with Badnavirus and Potyvirus degenerate primers and suggests their co-infection. insertsUSA) and were screened by DH5α standard cells colony were transformed PCR method. by Three heat shock method [25]. The positive colonies for expected size Bangalore, India) and consensus sequences were determined to Whereas, Encaster, Golden Girl, Golden Yellow Lucifer and clones for each canna sample were sequenced (Genei Pvt. Ltd., Badnavirus Red President showed thePotyvirus expected size amplifications with genus specific primers only. Kanchan cultivar eliminate any ambiguity before depositing to GenBank database. showed amplification with genus specific primers only. Sequence data of virus isolates was analyzed by BLASTn (http:// and respective sequences in database available worldwide. The We also checked the existence of CaYSV and TAV, by CaYSV and www.ncbi.nlm.nih.gov/BLASTn) and compared with the existing theirTAV specificabsence. primers, however, our several attempts failed to of isolates under study with selected virus sequences were produce any amplicon with these specific primers suggesting specific pairwise percent nucleotide and amino acid identity The amplicon from each cultivar was cloned and three virus isolates under study with our previously published isolates positive colonies, for each amplicon, were sequenced and the (highlightedcalculated by in ClustalW2grey shadowing) tool. The and phylogeneticother available relationship virus isolates of consensus sequence data obtained for each clone was submitted from India and abroad was also established using Molecular

to GenBank under different accessions (Table 2). BLASTn Evolutionary Genetics Analysis tool, MEGA v6.0, with 1000 analysis of accessions: KT447042 (from Allegheny cultivar), Ann Virol Res 2(1): 1008 (2016) 2/6 Raj et al. (2016) Email:

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Figure 1 repository at CSIR-NBRI. The healthy plant of each cultivar has also been shown as control. Leaves from ten different canna cultivars exhibiting streaks, mosaic accompanied with streaks, mottling, and severe mosaic symptoms gown in Canna

Table 1:

Symptoms and percent (%) disease incidence (based on visual symptoms) observed in canna repository at CSIR-NBRI, Lucknow, India Diseased/ healthy plants observed S.during No. JanuaryCultivars 2012 to January 2014. Symptoms Total % disease 2012 2013 2014 Allegheny Bengal Tiger 1. SM, Stk 2/12 3/17 5/21 10/50 20.0 2. SStk 24/127 31/141 30/232 85/500 17.0 Red President 3. Black Knight SM, Stk 29/67 45/96 56/137 130/300 43.3 M 4. Stk 36/109 41/134 48/157 125/400 31.2 SM 5. Kanchan 3/27 5/34 6/39 14/100 14.0 6. Eileen Gallo 2/19 3/23 7/38 12/80 15.0 7. New Delhi Yellow M, Stk 2/36 4/41 2/23 8/100 8.0 Encaster 8. Golden Girl M, SM, Stk 10/27 8/24 17/49 35/100 35.0 9. M, SM, Stk 9/32 14/35 17/53 40/120 33.3 Abbreviations: 10. Golden Yellow Lucifer M, SM, Stk 16/26 24/29 27/35 67/90 74.4 M: Mosaic; SM: Severe mosaic; Stk: Streak; SStk: Severe streak Table 2: S. No. Cultivar name CaYMV BYMV CMV Detail of GenBank accession numbers for CaYMV, BYMV, and CMV isolated from different canna cultivars. Allegheny Bengal Tiger 1 KT447042 KR922820 KP177963 2 KT447043 KP177962 ̶ 3 Black Knight KT447039 KT748759 JX570737 Encaster 4 Eileen Gallo KT447041 KP177961 KR922819 5 KJ561636 ̶ ̶ 6 Golden Girl KJ561637 ̶ ̶ 7 Golden Yellow Lucifer KJ561635 ̶ ̶ Red President 8 Kanchan ̶ KP177960 ̶ 9 KC688263 ̶ ̶ - = virus not detected 10 New Delhi Yellow ̶ ̶ ̶

KT447043 (Bengal Tiger), KT447039 (Black Knight), KT447041 and KP177960 (Kanchan) isolates showed 95-96% identity to (Eileen Gallo), KJ561635 (Golden Yellow Lucifer), KJ561636 each other and 77-97% identity with various strains of BYMV. (Encaster), KJ561637 (Golden Girl), and KJ561638 (Allegheny) Sequence analysis of JX570737 (Black Knight), KP177963 showed 98-99% nucleotide sequence identity to each other (Allegheny) and KR922819 (Eileen Gallo) 97-99% identity with and 93-99% identity with worldwide available CaYMV isolates. presence of CaYMV, BYMV, and CMV in Indian canna cultivars. CMV isolates of subgroup I. Molecular analyses confirmed the Sequence analysis of KR922820 (Allegheny), KP177962 (Bengal Tiger), KP177961 (Eileen Gallo), KT748759 (Black Knight) Ann Virol Res 2(1): 1008 (2016) 3/6 Raj et al. (2016) Email:

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Figure 2 Badnavirus Potyvirus Cucumovirus

Agarose gel image showing expected size bands of ~600 bp with (a), ~1500 bp with (b) and ~900 bp with (c) degenerate primers in PCR/RT-PCRs. Lanes 1-10: ten canna leaf samples from different cultivars (detailed in MM), Lane P: respective positive controls, Lane N: asymptomatic leaf sample, Lane M: 1 kb DNA ladder as size marker.

(a) EU377673: PBCErV, Australia 100 KP177960: BYMV isolate-AK3, India FJ439817: SBCV isolate BataviaD, West Indies (b) (c) 99 JX570737: CMV isolate-AK1, India KJ561637: CaYMV isolate-16, India 69 89 KJ561634: BYMV isolate-AK1, India 55 EF178298: CMV isolate-Gmt, India 83 KT447039: CaYMV isolate-18, India KJ561638: CaYMV isolate-17, India 58 KJ781366: BYMV isolate-AK2, India 42 AM158321: CMV isolate-UP, India 51 KT447041: CaYMV isolate-19, India HE774734: CaYMV isolate-PHC-C5, Thailand KR922820: BYMV isolate-AK-6, India 19 34 EF608461: CMV KPS10, Thailand SB-IB 45 HE774735: CaYMV isolate-PHC-51, Thailand 100 KP177961: BYMV isolate-AK4, India KT447043: CaYMV isolate-21, India 94 18 78 AY560556: CMV isolate-SG15, Thailand KJ561636: CaYMV isolate-15, India 67 KP177962: BYMV isolate-AK5, India EF156357: CaYMV isolate-1, Italy 50 DQ640743: CMV isolate-Mah, India 13 EF156358: CaYMV isolate-2, Italy KT748759: BYMV isolate-AK7, India 71 EF156359: CaYMV isolate-3, Italy 99 73 97 EF592169: BYMV isolate-Csz, China 99 AF350450: CMV isolate-H, India 10 KT447042: CaYMV isolate-20, India 97 FN994894: CaYMV isolate-JJ1, Thailand EF592168: BYMV isolate-Cgz, China FJ268746: CMV isolate-cah1, China 95 KC208485: CaYMV isolate-zz4, China KC195857: CaYMV isolate-zz2, China JQ012792: BYMV isolate-CJ2, India 47 D12499: CMV isolate-Y, Japan KC208484: CaYMV isolate-zz3, China 80 43 100 99 JQ178366: BYMV isolate-CJ1, India KJ561635: CaYMV isolate-14, India D00385: CMV isolate-O, Japan FN994895 CaYMV isolate-BK1nt, Thailand 99 21 34 HG970851: BYMV isolate-SP1, Australia 100 SB-IA 43 64 FN994892: CaYMV isolate-BK1, Thailand D10538: CMV isolate-Fny, USA 86 AF214005: BSV,Australia HG970854: BYMV isolate-GB32A, Australia 60 JF911397: BSV isolate Bo01, Japan 75 78 KR922819: CMV isolate-AK-3, India 75 JX570735: CaYMV isolate-5, India HG970852: BYMV isolate-GB17A, Australia 93 99 KC880353: CaYMV isolate-11, India HG970860: BYMV isolate-PN83A, Australia 98 KP177963: CMV isolate-AK-2, India 40 JX570733: CaYMV isolate-3, India 19 KF233987: CaYMV isolate-12, India KF632713: BYMV isolate-SW-9, Australia M21464: CMV isolate-Q, Australia KF233988: CaYMV isolate-13, India 33 18 HG970865: BYMV isolate-AR93C, Australia SB-II 19 JX570734: CaYMV isolate-4, India 99 L15336: CMV isolate-Trk, Hungary KC880352: CaYMV isolate-10, India 11 59 HG970869: BYMV isolate-NG-1, Australia 8 EF189148: CaYMV isolate-V17, Austria EF153735: TAV isolate Kolkata, India 51 JX228965: CaYMV isolate-1, India JQ686722: OrMV isolate-Glad-8, India OG 13 JX228966: CaYMV isolate-2, India NC_ 002040: PSV isolate-Er, China KC880351: CaYMV isolate-9, India AY994102: OrMV, New Zealand 28 0.1 43 KC688262: CaYMV isolate-7, India 0.1 11 KC688263: CaYMV isolate-8, India 0.1 32 KC688261: CaYMV isolate-6, India

Figure 3 with worldwide reported strains. The virus isolates reported herein and previous studies by our group are shown in bold. The evolutionary history Dendrograms showing close phylogenetic relationship of canna isolates of CaYMV (a), BYMV (b) and CMV (c) (highlighted in grey shadowing)

distanceswas inferred are using arbitrary. the Neighbor-Joining method conducted in MEGA v6.1. The percentage of replicate trees in which the associated taxas clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Horizontal distances are proportional to sequence distances, vertical

The phylogenetic relationships of our isolates with each other and with reported isolates of CaYMV, BYMV, and CMV relationshipsof BYMV sequences and clustered under with study Asian (KR922820, (Indian and KP177962, Chinese) KP177961, KT748759, and KP177960) revealed their close geneticwas assessed diversity using was MEGA observed tool in which all CaYMV suggest isolates a high and genetic they groupeddiversity into among two them main clusters.(Figure 3). The In CaYMV phylogeny, sequences significant under theisolates occurrence of BYMV of (Figureboth subgroup 3b). The IA phylogenetic and IB members analysis in canna with CMV sequences (JX570737, KP177963, and KR922819) revealed showedstudy (KT447042, close phylogenetic KT447043, relationships KT447039, together KT447041, and withKJ561635, other because CMV-AK1 isolate (JX570737) showed close phylogenetic KJ561636, KJ561637, and KJ561638) grouped in a cluster and relationships with subgroup IB isolates, while CMV-AK2 and -AK3 isolates (KP177963 and KR922819) showed close phylogenetic CaYMV isolates from India and abroad (Figure 3a). Phylogeny relationships with subgroup IA isolates (Figure 3c). Ann Virol Res 2(1): 1008 (2016) 4/6 Raj et al. (2016) Email:

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9. Rajakaruna P, Shafiekhani M, Kim T, Payton M, Chauhan R, Verchot CMVSince and 1985TAV of [14] genus to theBadnavirus recent report, Potyvirus of virus and infectionCucumovirus [9], plant viruses belonging to the genera Potyvirus, Cucumovirus and J. Production of discernible disease phenotypes in Canna by five cannas are known to be susceptible for CaYMV, CaYSV, BYMV, Badnavirus

BYMV isolates in few canna cultivars was reported by our group Canna yellow mottle virus in Canna from abroad [9-14,20]. In India, the detection of CaYMV and . Plant Pathol. 2014; 63: 821-830. 10. Pappu HR, Druffel KB, Eastwell KC. present[28,29]. in However, canna repository), the present rate of study disease was incidences, undertaken most with and spp. in Washington State. Plant Dis. 2008; 92: 1136. objectives to know the status of virus infection (which viruses are of Canna yellow mottle virus (CaYMV) in Italy and in The Netherlands. least prevalent viruses in natural condition in ten commercially 11. Marino MT, Ragozzino E, Lockhart BEL, Miglino R, Alioto D. First report germplasm preservation in CSIR-NBRI, India. The present report New Dis Reports. 2007; 15: 2. Canna yellow mottle important canna cultivars maintained in field condition for virus 12. Momol MT, Lockhart BEL, Dankers H, Adkins S. CMV) or individual virus infection in canna plants may provide detected in canna in Florida.Canna Plant yellow Health mottleProg. 2004. virus in North valuableof occurrence insight of regardingcomplexes the of threevirus virusesinfections (CaYMV, and the BYMV genome and 13. Lockhart BEL. Occurrence of America. Acta Hort. 1988; 234: 69-72. mayinformation. raise the The sincere co-existence concern ofto CaYMVthe canna and growers BYMV was in India. reported earlier [9]; however, the co-existence of CaYMV, BYMV and CMV 14. Yamashita S, Natsuaki T, Doi Y, Yora K. Canna yellow mottle virus, a CONCLUSION non-enveloped small-bacilliform virus in Canna sp. Jap. 1985; 51: 642- 646.Saidi A, Safaeizadeh M. First report of Cucumber mosaic virus on Canna Badnavirus, Potyvirus and Cucumovirus) in Canna species has been detected at molecular 15. Coexistence of three virus genera ( indica in Iran. Australas Plant Dis Notes. 2012; 7: 119-121. Report of Cucumber mosaic virus in Eryngium amethystinum, Canna 16. Fisher JR, Sanchez-Cuevas MC, Nameth ST, Woods VL, Ellett CW. First spp., and Aquilegia oflevel BYMV and identified and CaYMV by issequence reported analyses earlier of in the canna cloned from PCR/RT- India; however,PCR product the amplified occurrence by theirof CMV respective in canna primers. is being The reported existence for hybrids in Ohio. Plant Dis. 1997; 81: 1331. 17. Hollings M, Stone OM. Tomato aspermy virus. CMI0 AAB Descriptions ACKNOWLEDGEMENTSthe first time. of Plant Viruses Wallingford. 1971; 79. -terminal region of Bean yellow 18. Qin Z, Mao Q, Ding A, Jin L, Peng W, Du Z et al. Sequence characterization mosaic virus and molecular detection of the 3’ A. Kumari is thankful to University Grant Commission, New infecting canna. IEEE/IET Electronic Library. 2010; 978: Delhi, India for fellowship (19-06/BC11(i)EU-IV). 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Cite this article Kumari A, Raj R, Kumar S, Chauhan PS, Raj SK (2016) Coexistence of Three Virus Genera (Badnavirus, Potyvirus and Cucumovirus) in Canna Species in India. Ann Virol Res 2(1): 1008.

Ann Virol Res 2(1): 1008 (2016) 6/6