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.

• Canna

• Canna yellow mottle virus

• Bean yellow mosaic virus

• Cucumber mosaic virus

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.

CaYMV: Canna yellow mottle virus; BYMV: Bean yellow mosaic virus; CaYSV: Canna yellow streak virus; CMV: Cucumber Mosaic Virus; TAV: Tomato Aspermy Virus; PCR: Polymerase Chain Reaction; RT-PCR: Reverse Transcription-Polymerase Chain Reaction, TEM: Transmission Electron Microscopy.

Cannas (Canna spp., family Cannaceae) are perennial plants grown in tropical and sub-tropical countries including India. They are largely used in borders of beds for their colourful foliages and flowers, besides being grown in lawns, parks, and other public places. Therefore, canna has significance due to their large shiny foliage and attractive flowers of red, yellow, orange, pink colour or any combinations of these colours [1]. Canna rhizome is consumed as food being rich in starch [2,3] and is also used for bio-ethanol production [4]. In recent years, cannas have got attention of breeders and floriculturists to develop new commercially important varieties [5].

Canna is reported to be susceptible to many phytopathogens such as: Xanthomonas cannae [6], Cordana veriscolor [7] Puccinia thaliae [8]. Besides this, Canna species are known as host of viruses worldwide. Canna yellow mottle virus (CaYMV) is reported to infect several canna species and is commonly associated with leaf mottling symptoms [9-14]. Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV) are known to infect canna and induces mosaic and streaks symptoms [9,15-17]. Bean yellow mosaic virus (BYMV), previously known as Canna mottle virus [13], is also reported to infect canna [9,18]. Canna yellow streak virus (CaYSV) has been reported in canna causing yellow streak disease [19,20]. The infection of viruses of single genus is common in canna, while co-infection of two different genera has also been reported [9,21], however, the coexistence of three viruses of different genera is not known in India. Here we report the infection of CaYMV, CMV, and BYMV in ten important Canna cultivars maintained in canna repository of CSIR-NBRI, India. Three (Allegheny, Black Knight, and Eileen Gallo) cultivars, out of ten, were co infected by CaYMV, CMV and BYMV. Knowledge of virus infection in canna may aid in their early detection, searching of virus-free propagating material or disease management.

Three consecutive surveys in January month of 2012, 2013, and 2014 were conducted for scoring the virus like diseases in ten different canna cultivars (Allegheny, Bengal Tiger, Black Knight, Red President, Kanchan, Eillen Gallo, New Delhi Yellow, Golden Girl, Encaster and Golden Yellow Lucifer) maintained at repository of CSIR-NBRI, Lucknow, India (latitude: 26° 51’N and longitude: 80o 55’E). Disease incidence in these ten cultivars was assessed by visual observation of symptoms. The symptomatic and asymptomatic (control) leaf samples were collected and used for virus detection.

For morphological virus identification, transmission electron microscopic (TEM) study was conducted using leaf dip preparations as described earlier [21]. The virus particles on grid were negatively stained with 2% Uranyl acetate and observed in a TEM (Philips EM420, USA) at 35-100 K magnification.

For molecular detection of viruses, a total of 30 symptomatic canna leaf samples from ten cannas cultivars (three samples from each cultivar) were subjected for PCR/RT-PCR. The total DNA and RNA were isolated independently from leaf samples using GenElute Plant Genomic DNA Miniprep kit (Sigma-Aldrich, MO, USA) and RNeasy Plant Mini Kit (Sigma-Aldrich, MO, USA), respectively following the manufacturer’s instructions and used as templates for PCR and RT-PCR. The DNA preparations were directly subjected for PCR with Badnavirus degenerate primers [22] following their recommended amplification conditions which expected to direct the amplification of ~600 bp band. The RNA preparations were reverse transcribed into cDNA with Potyvirus [23] and Cucumovirus [24] downstream degenerate primers independently using GoScript™ Reverse Transcription System (Promega Corp., Madison, USA) following their instructions. Further, PCR were conducted following the conditions described earlier for Potyvirus [23] and Cucumovirus [24] detection, respectively in a SureCycler 8800 Thermal Cycler (Agilent Technologies, USA). The sizes of products were assessed on 1% agarose gel with 1 kb DNA size marker (Thermo-Fisher Scientific Inc., Pittsburgh, USA).

The expected size PCR products from the gel were eluted using Wizard® SV Gel and PCR Clean-Up System (Promega Corp., Madison, USA). The purified products were cloned separately into pGEM®-T Easy Vector System-I (Promega Corporation, Madison, USA) and Escherichia coli DH5α cells were transformed by heat shock method [25]. The positive colonies for expected size inserts were screened by standard colony PCR method. Three clones for each canna sample were sequenced (Genei Pvt. Ltd., Bangalore, India) and consensus sequences were determined to eliminate any ambiguity before depositing to GenBank database. Sequence data of virus isolates was analyzed by BLASTn (http:// www.ncbi.nlm.nih.gov/BLASTn) and compared with the existing and respective sequences in database available worldwide. The specific pairwise percent nucleotide and amino acid identity of isolates under study with selected virus sequences were calculated by ClustalW2 tool. The phylogenetic relationship of virus isolates under study with our previously published isolates (highlighted in grey shadowing) and other available virus isolates from India and abroad was also established using Molecular Evolutionary Genetics Analysis tool, MEGA v6.0, with 1000 replicates bootstrapping [26]. The dendrogram was generated employing Neighbour-Joining (N-J) method and viewed by the N-J plot program of MEGA tool. Pineapple bacilliform erectifolius virus (PBCErV, EU377376) and Sugarcane bacilliform virus (SBCV, FJ439817) strains have been used as out group members for rooting of the phylogenetic tree.

During surveys (January 2012-2014) of canna repository, mild to severe symptoms were observed on leaves of Allegheny, Bengal Tiger, Black Knight, Red President, Kanchan, Eileen Gallo, New Delhi Yellow, Golden Girl, Encaster and Golden Yellow Lucifer cultivars (Figure 1).

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

Visual observation of symptoms in these cultivars revealed 8.0 to 74.4% disease incidences (Table 1).

TEM analysis of infected Allegheny, Black Knight, and Eileen Gallo samples revealed the presence of typical bacilliform, flexuous rods and cored spherical virions measuring about 130x30 nm, 600x11 nm, and 28 nm (in diameter), respectively and suggest their co infection. Bengal Tiger cultivar revealed the mixed infection of bacilliform and flexuous rod shaped virus particles. The four cultivars: Encaster, Golden Girl, Golden Yellow Lucifer and Red President showed the presence bacilliform virus particles and suggest badnavirus infection only, while Kanchan cultivar showed the presence of flexuous rod shaped virus particles only and suggest potyvirus infection.

In PCR/RT-PCR analyses, 22 samples showed expected size amplification of ~600 bp with Badnavirus degenerate primers from Allegheny, Bengal Tiger, Black Knight, Eileen Gallo, Encaster, Golden Girl, Golden Yellow Lucifer and Red President cultivars. The 9 samples showed amplification of expected size (~1600 bp) with Potyvirus degenerate primers from Allegheny, Bengal Tiger, Black Knight, Eileen Gallo, Encaster and Kanchan cultivars, whereas, 5 samples showed expected size ~900 bp amplicon with Cucumovirus genus specific primers from Allegheny, Black Knight and, Eileen Gallo cultivars. A representative figure has been shown to display the obtained amplicons from ten canna cultivars (Figure 2a-c).

Figure 2 Agarose gel image showing expected size bands of ~600 bp with Badnavirus (a), ~1500 bp with Potyvirus (b) and ~900 bp with Cucumovirus (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.

All the three leaf samples of New Delhi Yellow cultivar failed to produce any band with any of these degenerate primers. Molecular analysis confirmed why no virus particles could be observed in New Delhi Yellow cultivar during TEM. Three cultivars: Allegheny, Black Knight and Eileen Gallo produced expected size amplicons with all three genus specific degenerate primers and suggest triple infection in them. Bengal Tiger cultivar also showed the amplicons with Badnavirus and Potyvirus degenerate primers and suggests their co-infection. Whereas, Encaster, Golden Girl, Golden Yellow Lucifer and Red President showed the expected size amplifications with Badnavirus genus specific primers only. Kanchan cultivar showed amplification with Potyvirus genus specific primers only. We also checked the existence of CaYSV and TAV, by CaYSV and TAV specific primers, however, our several attempts failed to produce any amplicon with these specific primers suggesting their absence.

The amplicon from each cultivar was cloned and three positive colonies, for each amplicon, were sequenced and the consensus sequence data obtained for each clone was submitted to GenBank under different accessions (Table 2).

BLASTn analysis of accessions: KT447042 (from Allegheny cultivar),KT447043 (Bengal Tiger), KT447039 (Black Knight), KT447041 (Eileen Gallo), KJ561635 (Golden Yellow Lucifer), KJ561636 (Encaster), KJ561637 (Golden Girl), and KJ561638 (Allegheny) showed 98-99% nucleotide sequence identity to each other and 93-99% identity with worldwide available CaYMV isolates. Sequence analysis of KR922820 (Allegheny), KP177962 (Bengal Tiger), KP177961 (Eileen Gallo), KT748759 (Black Knight)and KP177960 (Kanchan) isolates showed 95-96% identity to each other and 77-97% identity with various strains of BYMV. Sequence analysis of JX570737 (Black Knight), KP177963 (Allegheny) and KR922819 (Eileen Gallo) 97-99% identity with CMV isolates of subgroup I. Molecular analyses confirmed the presence of CaYMV, BYMV, and CMV in Indian canna cultivars.

The phylogenetic relationships of our isolates with each other and with reported isolates of CaYMV, BYMV, and CMV was assessed using MEGA tool which suggest a high genetic diversity among them (Figure 3).

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

In phylogeny, significant genetic diversity was observed in all CaYMV isolates and they grouped into two main clusters. The CaYMV sequences under study (KT447042, KT447043, KT447039, KT447041, KJ561635, KJ561636, KJ561637, and KJ561638) grouped in a cluster and showed close phylogenetic relationships together and with other CaYMV isolates from India and abroad (Figure 3a). Phylogeny of BYMV sequences under study (KR922820, KP177962, KP177961, KT748759, and KP177960) revealed their close relationships and clustered with Asian (Indian and Chinese) isolates of BYMV (Figure 3b). The phylogenetic analysis with CMV sequences (JX570737, KP177963, and KR922819) revealed the occurrence of both subgroup IA and IB members in canna because CMV-AK1 isolate (JX570737) showed close phylogenetic relationships with subgroup IB isolates, while CMV-AK2 and -AK3 isolates (KP177963 and KR922819) showed close phylogenetic relationships with subgroup IA isolates (Figure 3c).

Since 1985 [14] to the recent report of virus infection [9], cannas are known to be susceptible for CaYMV, CaYSV, BYMV, CMV and TAV of genus Badnavirus, Potyvirus and Cucumovirus from abroad [9-14,20]. In India, the detection of CaYMV and BYMV isolates in few canna cultivars was reported by our group [28,29]. However, the present study was undertaken with objectives to know the status of virus infection (which viruses are present in canna repository), rate of disease incidences, most and least prevalent viruses in natural condition in ten commercially important canna cultivars maintained in field condition for germplasm preservation in CSIR-NBRI, India. The present report of occurrence of complexes of three viruses (CaYMV, BYMV and CMV) or individual virus infection in canna plants may provide valuable insight regarding the virus infections and the genome information. The co-existence of CaYMV and BYMV was reported earlier [9]; however, the co-existence of CaYMV, BYMV and CMV may raise the sincere concern to the canna growers in India.

Coexistence of three virus genera (Badnavirus, Potyvirus and Cucumovirus) in Canna species has been detected at molecular level and identified by sequence analyses of the cloned PCR/RTPCR product amplified by their respective primers. The existence of BYMV and CaYMV is reported earlier in canna from India; however, the occurrence of CMV in canna is being reported for the first time.

A. Kumari is thankful to University Grant Commission, New Delhi, India for fellowship (19-06/BC11(i)EU-IV). Thanks are due to the Director, CSIR-NBRI, Lucknow for facilities to accomplish this work.

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