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Badnavirus, Potyvirus and Cucumovirus) in Canna Species in India

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Badnavirus, Potyvirus and Cucumovirus) in Canna Species in India Central Annals of Virology and Research Bringing Excellence in Open Access 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 areis commonlyknown 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 maintained different genera in canna is not repository known in India.of CSIR-NBRI, Here we reportIndia. 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: 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 erectifoliusgenerated 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 assessedrepository 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 measuringcultivar 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 totalPCR/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 thefor 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] RNAfollowing preparations their wererecommended 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 Redfrom 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
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