Sequence characterization of Dasheen mosaic virus ... - Springer Link

Arch Virol (2009) 154:159–162 DOI 10.1007/s00705-008-0257-9

ANNOTATED SEQUENCE RECORD

Sequence characterization of Dasheen mosaic virus isolates from cocoyam in Nicaragua Guillermo Reyes Æ Jon N. E. Ramsell Æ Marie Nyman Æ Anders Kvarnheden

Received: 6 August 2008 / Accepted: 21 October 2008 / Published online: 26 November 2008 Ó Springer-Verlag 2008

Abstract Dasheen mosaic virus (DsMV) is an important constraint to production of cocoyam (Xanthosoma spp.) in Nicaragua. Reverse transcription polymerase chain reaction was used to amplify the coat protein (CP) region from ten Nicaraguan DsMV isolates. These isolates showed high nucleotide identity to DsMV isolates from the USA, eastern Asia and Australasia. All Nicaraguan isolates except one shared a tandem repeat in the N-terminus of the CP. Phylogenetic analyses showed that the Nicaraguan isolates formed two distinct subgroups correlated with geographic

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AM910398–AM910407.

Electronic supplementary material The online version of this article (doi:10.1007/s00705-008-0257-9) contains supplementary material, which is available to authorized users. G. Reyes J. N. E. Ramsell M. Nyman A. Kvarnheden (&) Department of Plant Biology and Forest Genetics, Uppsala BioCenter SLU, Box 7080, 750 07 Uppsala, Sweden e-mail: [email protected] G. Reyes Universidad Nacional Agraria, Box 453, Managua, Nicaragua Present Address: J. N. E. Ramsell Section for Virology and Serology, Department of Animal Health, National Veterinary Institute, Box 750, Sentrum, 0106 Oslo, Norway Present Address: M. Nyman The Swedish Gene Technology Advisory Board, Retzius va¨g 13A, 171 77 Stockholm, Sweden

origin. This can be explained by different origins of the cocoyam genotypes grown in these regions.

Cocoyam (Xanthosoma spp.) is a crop cultivated in many tropical and subtropical countries for its edible leaves, corm and cormels. It belongs to the family Araceae and originated in tropical and southern subtropical America [6]. Of all root and tuber crops grown in Nicaragua, cocoyam is the most important export crop, and the second most important regarding production area [5]. In recent years, the total national production area and yield have significantly decreased, mainly due to diseases that are disseminated through planting material. Disease caused by Dasheen mosaic virus (DsMV) and root rot disease are currently the most important diseases constraining cocoyam production. Flowering in cocoyam is rare, and when it occurs, seeds are mostly nonviable. Hence, cocoyam has to be propagated through somatic portions of corms and cormels, and this favors spread of DsMV-infected plant material and subsequent disease. The virus can be also transmitted by several winged aphid species, which are effective vectors of DsMV [15]. In Central America, DsMV was first identified affecting cocoyam in Costa Rica based on symptoms and electron microscopy [9]. In Nicaragua, the virus infects 68–100% of the plants in commercial plantations [11], and the yield losses by the virus are estimated to be around 25% [12]. DsMV is thought to be the most important viral pathogen attacking wild and cultivated species of the family Araceae worldwide [2, 10, 13], especially in tropical and subtropical countries. It was first described in 1970 [20] and is classified as a member of the genus Potyvirus, family Potyviridae [1, 14]. The genome of

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DsMV consists of linear positive-sense single-stranded (ss)RNA, which contains a single open reading frame, flanked by a non-translated region at both (50 and 30 ) ends [2]. DsMV has flexuous filamentous particles ([700 nm) and is transmitted by aphids in a non-persistent manner [19]. In recent years, the number of DsMV sequences reported in the literature has increased. Partial sequences from Colocasia [2, 7, 8], Zantedeschia [2], Pinellia [13], and Caladium [4] as well as one complete genome sequence (9,991 nt) from Zantedeschia [2] have been reported. Phylogenetic analyses have shown that DsMV belongs to the Bean common mosaic virus (BCMV) group within the genus Potyvirus and that DsMV is most closely related to Vanilla mosaic virus (VanMV) [1]. Recently, VanMV was suggested to be a strain of DsMV that exclusively infects vanilla [3]. However, no sequences of DsMV isolates from Central America or from Xanthosoma have so far been reported. Therefore, the objective of this study was to determine the partial sequences of Nicaraguan DsMV isolates from cocoyam and to study their relationships to other isolates of DsMV and VanMV. Leaf samples from three cocoyam genotypes (Blanco, Nueva Guinea and Masaya) with visual symptoms of DsMV infection were collected from four commercial plantations in the two most important cocoyam-producing regions in Nicaragua (Table 1). The samples were brought fresh to the Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Sweden, and kept at -20°C until analysis. Pieces of symptomatic leaves were ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted from the homogenate using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed using OneStep RT-PCR (Qiagen) according to the manufacturer’s instructions. Specific primers I and II [4] were used to amplify a 1.2-kb fragment containing the

partial CP region and 30 -untranslated region (UTR) of DsMV for the isolates NiNG1, NiNG3, NiNG4 and NiNG18. Forward primer P2F (50 AGG TTG TAT TGC AGG CAG ATG 30 ) and reverse primer P2R (50 GCC AAT AAC TGT GGC CTG TT 30 ) were designed based on the conserved genomic regions for DsMV isolates available in NCBI GenBank (http://www.ncbi.nlm.nih.gov). These primers were used to amplify partial CP and 30 -untranslated regions (1.0 kb), corresponding to nucleotides (nt) 8,795– 9,993 in DsMV-M13 [2] (accession number AJ298033) for the isolates NiNG67, NiMY70, NiMY72, NiMY75, NiMY76 and NiMY78. The amplification protocol consisted of a 30 min initial reverse transcription step at 50°C, followed by 15 min at 95°C to initiate the PCR. The samples were then subjected to 40 cycles of 1 min at 94°C, 1 min at 41°C and 1 min at 72°C and then kept 10 min at 72°C for a final extension step. As negative controls, PCR was run with samples from non-infected cocoyam plants and with water. The PCR products were separated by electrophoresis on 1.4% agarose gels containing 0.59 TBE buffer. The bands of the expected size were subsequently purified using a QIAquick Gel Extraction Kit (Qiagen). The purified amplicons were cloned into pGEM-T Easy Vector (Promega, Madison, WI, USA) and transformed into Escherichia coli DH5a Competent Cells (Life Technology, Gibco, Lexington, USA) according to the manufacturer’s instructions. Recombinant clones were sequenced using the DYEnamicTM Terminator Cycle Sequencing Premix Kit (Amersham Pharmacia, Little Chalfont, England) and an ABI PRISMTM 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, CT). At least two clones per isolate were sequenced. The sequence for one clone of each isolate was determined on both strands. Nucleotide and predicted amino acid (aa) sequences were aligned, analysed and compared with other DsMV isolates that were available in GenBank

Table 1 Description of Nicaraguan DsMV isolates from cocoyam in this study Proposed name

Genotype

Region

Acc. No.

DsMV-NiNG1

Blanco

Nueva Guinea (Rain forest)

AM910398

DsMV-NiNG3

Blanco


AM910399

DsMV-NiNG4

Blanco


AM910400

DsMV-NiNG67

Blanco


AM910401

DsMV-NiNG18

Nueva Guinea


AM910402

DsMV-NiMY70

Masaya

Masaya (Central Pacific)

AM910403

DsMV-NiMY72

Masaya


AM910404

DsMV-NiMY75

Masaya


AM910405

DsMV-NiMY76

Masaya


AM910406

DsMV-NiMY78

Masaya


AM910407

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(Supplementary Table 1) using Clustal W [17]. Phylogenetic analysis was carried out using Phylogenetic Analysis Using Parsimony (PAUP*) software, version 4.0 Beta [16]. The distances matrices for the neighbour-joining analyses were calculated using the Kimura two-parameter model. The results obtained from the neighbour-joining analyses were further assessed by maximum parsimony analysis. Essentially the same topologies were obtained with both methods. The robustness of the internal branches of the tree was estimated by bootstrap analysis using 1,000 replicates. Cocoyam plants of three genotypes with leaf distortion, vein chlorosis or mosaic feathering along the veins were collected from four commercial plantations in the regions Nueva Guinea and Masaya (Table 1). RT-PCR and two sets of DsMV primers allowed the amplification of the expected cDNA fragments (1.0 and 1.2 kb) from 25 of 27 tested samples. Partial CP sequences (975 nt, beginning with the DAG motif) were determined for ten Nicaraguan DsMV isolates from cocoyam. Sequence comparisons among the Nicaraguan isolates showed that the nt and aa sequence identities varied from 84 to 100% and from 94 to 100%, respectively (Supplementary Table 2). The isolates from the region Masaya (NiMY70, NiMY72, NiMY75, NiMY76, and NiMY78) all shared high sequence identities (99% at the nt and aa level). Isolates NiNG1, NiNG3, NiNG4 and NiNG67 (from the region Nueva Guinea) showed 96– 100% nt and aa identities to each other. NiNG18 was the most divergent of the NiNG isolates and displayed 87–88% nt identity to the other NiNG isolates and 84% nt identity to NiMY isolates. Comparison of the Nicaraguan isolates with previously published DsMV isolates (Supplementary Table 1) showed that NiNG1, NiNG3, NiNG4 and NiNG67 shared the highest nt identity (96–98%) with DsMV-LA from Colocasia in Florida [8]. Also for NiNG18, the nt identity was highest to DsMV-LA (86%). The NiMY isolates shared 87–90% nt identity with DsMV-LA and DsMV-DK, isolated from Japanese Colocasia [2]. In the N-terminus of the CP, all Nicaraguan isolates except NiNG18 shared a 10-aa sequence repeated three times in tandem (Supplementary Fig. 1). This GNNTNTNT(N/S)T sequence repeat has previously been reported to be present in DsMV-LA [7, 8]. NiNG18 showed a 19-aa-long deletion, which included the first 10aa repeat. Differences in length of the CP coding region is a common characteristic in potyviruses [14], and this has been reported also for DsMV [2, 4, 13]. The aa triplet DAG, related to aphid transmission [14], was present in all Nicaraguan DsMV isolate sequences and in all isolates from GenBank, except for DsMV-Ch (isolated from Caladium in USA) [4], where DAG is changed to DAR (Supplementary Fig. 1).

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100 DSMV-NiNG1 86 DsMV-NiNG4 60 DsMV-LA 100 DsMV-NiNG67 DsMV-NiNG3 DsMV-NiNG18 DsMV-NiMY70 54 DsMV-NiMY75 DsMV-NiMY76 100 DsMV-NiMY78

100

67

DsMV-NiMY72 DsMV-DK DsMV-YN80

62

94

DsMV-Ch DsMV-M13 DsMV-ND

56 59 59

60

DsMV-S

64

DsMV-TEN DsMV-TW

62 72

DsMV-TW3 DsMV-VN/Ce2 DsMV-VN/Ce1 DsMV-VN/Tt1

94

DsMV-1

96

DsMV-2 VaMV-FP VaMV-CI-AT

100 59

DsMV-SY1 100 100

ZYMV-Singapore ZYMV-B ZYMV-H272-8

0.05 substitutions/site

Fig. 1 Neighbour-joining analysis showing predicted relationships between Dasheen mosaic virus (DsMV) isolates based on the alignment of 975 nt of the coat protein region. Numbers represent the bootstrap values (%) out of 1000 replicates. Only bootstrap values higher than 50 are shown. Horizontal lines are in proportion to the number of nucleotide differences between branch nodes. The tree was rooted with Zucchini yellow mosaic virus (ZYMV) isolates as outgroup. The origin and accession numbers of virus isolates are given in Table 1 and Supplementary Table 1

Phylogenetic analysis using partial nt sequences for the CP coding region of Nicaraguan DsMV isolates (975 nt), previously reported DsMV isolates, Zucchini yellow mosaic virus (ZYMV) and VanMV isolates (Fig. 1) showed that the DsMV and VanMV isolates formed a monophyletic group (bootstrap value 100%). The Nicaraguan isolates were placed within the DsMV/VanMV clade, confirming that they belong to this species. The Nicaraguan DsMV isolates formed two subgroups: one subgroup with the NiMY isolates and another subgroup with the NiNG isolates. The NiMY subgroup was well supported (bootstrap value 100%), and these isolates showed a very close relationship. Except for NiNG18, there was also a strong support in the analysis for the NiNG subgroup (bootstrap value 99%). The phylogenetic analysis placed DsMV-LA in the NiNG subgroup.

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DsMV appears to have a worldwide distribution and infects many different species within the family Araceae [2, 19]. However, there is still limited knowledge, including sequence information, about this virus. By RT-PCR, we have confirmed the association between disease symptoms and DsMV infection in cocoyam. In a previous study [11], the presence of DsMV in plants with the symptoms described above was corroborated by enzyme-linked immunosorbent assay (ELISA). The positive RT-PCR tests further supported the correlation between disease symptoms and DsMV infection. The sequence analyses showed the close relationship between the DsMV isolates from cocoyam and isolates from other aroids. The Nicaraguan cocoyam isolates formed two distinct subgroups correlated with geographic origin. In Nicaragua, located in the center of origin for the genus Xanthosoma, cocoyam is one of the most ancient crops [18]. Until late 1970s, cocoyam production in the traditional production areas was devoted to national consumption. In the beginning of the 1980s, Nueva Guinea and Blanco cocoyam genotypes were introduced from Costa Rica into the south Caribbean rainforest region, where most of the production is now devoted to export. Currently, the cocoyam production relies on three main cocoyam genotypes: Nueva Guinea and Blanco, grown in the rainforest region, and Masaya in the central Pacific area. The distinct origin of the cocoyam genotypes is the possible cause of the genetic differences between virus isolates from the NiNG and NiMY subgroups. The fact that DsMV-LA was isolated at a location geographically close to Nicaragua, (Florida, USA) could explain the close genetic relationship between the NiNG isolates and DsMV-LA. To obtain a more complete picture of the diversity among isolates of DsMV, additional isolates from different geographic origins and hosts should be sequenced and studied. Acknowledgments The authors wish to thank UNA-SLU PhD Program for its collaborative efforts. This study was funded by The Swedish International Development Cooperation Agency (Sida/SAREC). We acknowledge the valuable collaboration of Ena Rivers Carcache during the sample collection.

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