Complete genome sequence of a potyvirus infecting yam beans (Pachyrhizus spp.) in Peru.

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1 Arch Virol (2012) 157: ; DOI /s Complete genome sequence of a potyvirus infecting yam beans (Pachyrhizus spp.) in Peru. Segundo Fuentes, Bettina Heider, Ruby Carolina Tasso, Elisa Romero, Thomas zum Felde and Jan Kreuze* International Potato Center, Apartado 1558, Lima 12, Peru * Corresponding author: j.kreuze@cgiar.org; tel: ; fax: Abstract In 2010, yam beans in a field trial in Peru showed viral disease symptoms. Graft-transmission and positive ELISA results using potyvirus specific antibodies suggested that symptoms could be the result of a potyviral infection. Small interfering RNA (sirna) were extracted from one of the samples and sent for high throughput sequencing. The full genome of a new potyvirus could be assembled from the resulting sirna sequences which was sufficiently different from other sequences to be considered a new species, which we have designated Yam bean mosaic virus (YBMV). Sequence similarity suggests YBMV was also detected in yam beans in Indonesia. Legume root and tuber crops play an important role in providing protein- and micro-nutrient-rich food for human consumption [2]. Yam beans (Pachyrhizus spp.) show by far the widest adaptation and the highest yield potential amongst legumes [6]. Native to Central and South America, Pachyrhizus spp. are also grown in Asia and parts of Africa. The genus comprises three cultivated species: Pachyrhizus ahipa, P. erosus, and P. tuberosus that produce large storage roots with high protein (up to 12 % in dry matter), and moderate zinc (up to 14 ppm) and iron content (up to 52 ppm; CIP, unpublished data). Given the good adaptation of genus Pachyrhizus to a wide range of environmental conditions and its ability to be productive in marginal production systems, yam beans possess a yet untapped potential to provide improved food quality and quantity while generating new income options and improving farming systems sustainability. The International Potato Center (CIP)

2 holds the most comprehensive collection of Pachyrhizus spp. in the world, and is implementing an initiative to unleash the full potential of the American yam bean. The maintenance and distribution of healthy plant genetic resources are of major importance in gene bank management. Moreover, virus diseases are important constraints to root and tuber crops, particularly in the tropics. Sørensen [7] attributed 20 40% of yield decrease in P. erosus to virus diseases. Despite these negative impacts on crop performance there is little information on virus diseases in yam bean species so far. Sørensen [7] observed two virus diseases, caused by Sincama mosaic virus (SMV) and Bean common mosaic virus (BCMV), in four of the five Pachyrhizus species, especially in fields bordering wild vegetation. Only P. ferrugenensis appeared to show some resistance. In his study, Sørensen [7] concluded that BCMV might become a serious problem in yam bean producing areas. Indeed, Damayanti et al. [1] also reported the occurrence of a strain of BCMV causing considerable damage on P. erosus in different locations in Indonesia. Partial 3 sequence of that virus indicated it was a distinct strain of BCMV which they designated BCMV-IYbn. Towards the end 2010, viral disease symptoms (mosaic with or without leaf deformation) were observed in a yam bean field trial at the CIP research station in San Ramon; Junín, Peru (wet tropics, 800 masl).. The disease could be graft transmitted and based on symptoms [1], an infection by the potyvirus BCMV was suspected. Plants were tested by NCM-ELISA using potyvirus specific antibodies (Agdia CAB 27200) to which all symptomatic plants reacted positively, whereas symptomless greenhouse grown plants reacted negative. Small interfering RNA (sirna) sequencing (Provider: Fasteris Life Sciences SA, Plan-les-Ouates, Switzerland) and assembly was performed on a sample (P. tuberosus accession CIP209014) showing severe mosaic and leaf deformation as described by Kreuze et al. [5], except that the reads were assembled using Velvet s AssemblyAssembler (v1.1). Original sirna reads can be found at: The full genome sequence of a virus (9635 nt excluding the poly A tail) could be assembled at an average nucleotide coverage of 604 fold and was deposited in GenBank (accession number JN190431). Viral sirna reads were then mapped back to the assembled viral genome using MAQ v0.6.6 ( and coverage was visualized using a custom script (available from the authors upon request). A total of reads between nts were obtained of which (34%) corresponded to YBMV. sirna reads were distributed relatively homogenously over the viral genome with a number strong peaks at various intervals. Viral sirna corresponded mostly to 21nt sirna followed by 22 nt and about 60% of reads were of positive strand polarity. Total small RNA size distribution showed strong dominance of 21nt RNAs similar to

3 what has been observed in other severe potyviral infections [5]. Pairwise sequence comparison and phylogenetic analysis using complete genomes of related potyviruses (Fig. 1A) showed that the identified virus was most closely related to BCMV (strains NL1, NL4, RU1, Blackeye; isolates MS1 and cowpea Y and R) with 71.3% nt identity. This is below the potyvirus species demarcation limit of 76% [4] and should therefore be considered a new virus species for which we have proposed the name Yam bean mosaic virus (YBMV) which is currently in the process of ratification by the ICTV. Further comparison to partial sequences available from GenBank identified with 96.6% nucleotide identity the fragment corresponding to the kb of a potyvirus from yam bean in Indonesia (accession number AB289438) [1], indicating it is the same virus as the one identified in this study. On the other hand two virus sequences isolated from black bean (Phaseolus vulgaris; DQ925422) and yard-long bean (Vigna unguiculata; DQ925425) in Vietnam [3] show % nt and 92% aa identity to YBMV over the CP encoding region, which would suggest these also represent (strains of) the same virus [4]. Phylogenetic analysis using only the CP region and including the identified related viruses showed they formed a well supported clade, separate from BCMV (Fig 1B) with CP nt and aa similarities of % and %. AB289438, DQ and DQ were all originally reported as isolates of BCMV [1, 3], probably because the CP nt similarity with other BCMV isolates was around the potyvirus demarcation limit of 76%. On the other hand the YBMV isolate from Indonesia did not react with BCMV specific antibody [1]. The YBMV isolate from Indonesia was shown to be mechanically and aphid transmitted and preliminary results also suggested seed transmission [1]. Presence of YBMV and related viruses in Peru, Indonesia and Vietnam in yam bean, black bean and yard-long bean indicates the occurrence of this virus in geographically distant locations and in different crops. It is possible that BCMV reported by Sørensen [7] may also be a misidentification and correspond to YBMV, in which case the virus may already have a worldwide distribution. In experimental inoculations performed by Damayanti et al. [1], its host range included species from the families Amaranthaceae (Gomphrena globosa), Leguminoseae (P. vulgaris, V. sinensis & Pisum sativum) and Solanaceae (Solanum lycopersicon). The occurrence of this new virus disease might present a considerable threat to the recently started and, so far, promising yam bean production in Africa as well as in South America. Future research should address the development of specific detection methods and asses the level of seed transmission in order to develop and implement appropriate phytosanitary measures to prevent further spread of the disease. Worldwide occurrence, variability and susceptibility and impact on yield of the various yam bean species represent other key parameter needed to asses the threat of this virus to yam bean production in the world.

4 Acknowledgements We would like to thank Mirella Flores for help with bioinformatics. References 1. Damayanti TA, Susilo D, Nurlaelah S, Sartiami D, Okuno T, Mise K (2008) First report of Bean common mosaic virus in yam bean [Pachyrhizus erosus (L.) Urban] in Indonesia. J Gen Plant Pathol 74: FAO (1979) Review on agriculture and development. Conventional crops imperil good protein. Ceres 12: Ha C, Revill P, Harding RM, Vu M, Dale JI (2008) Identification and sequence analysis of potyviruses infecting crops in Vietnam. Arch Virol 153: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (2011) Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on the Taxonomy of Viruses. San Diego, CA: Elsevier Academic Press 5. Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388: NRC (1979) Tropical legumes. Resources for the future. National Academy of Science, Washington, DC, USA, NRC National Research Council 7. Sørensen M (1996) Yam bean (Pachyrhizus DC.) Promoting the conservation and use of underutilized and neglected crops. 2. Institute of Plant Genetics and Crop Plant Research (IPK), International Plant Genetic Resources Institute (IPGRI), Gatersleben, Germany; Rome, Italy. Figure 1. Phylogenetic tree of complete genome sequences (9635 nt ; A) and CP region (828 nt; B) of Yam bean mosaic virus (YBMV) and related viruses. YBMV shows only 71.3% nt sequence identity to Bean common mosaic virus (BCMV) over the complete genome indicating a new species (species demarcation: 76%). On the other hand partial sequence comparison in (B) indicates a virus identified as a strain of BCMV in yam bean in Indonesia also corresponds to YBMV. Analysis was performed using the MEGA5 program. Complete potyvirus genomes were downloaded from the

5 NCBI database and aligned using the Clustal W algorithm. A tree was then generated using neighbour-joining with the maximum composite likelihood method and 1000 bootstrap replicates, and branches corresponding to recognized viruses were collapsed into triangles. Sequences used in analysis: A) Soybean mosaic virus FJ FJ640959, FJ FJ640972, FJ FJ640982, HM590054, HM590055, HQ166266, HQ & HQ166265; Watermelon mosaic virus EU EU660590, DQ399708, NC & AB218280; Calla lily latent virus EF EU105299; Wisteria vein mosaic virus AY656816; Fritilary virus Y AM039800; East Asian passiflora virus AB246773; Peanut stripe virus U34972, U05771 & AY968604; Bean common mosaic virus EU761198, AY112735, DQ666332, GQ219793, AY863025, AJ312438, AY575773, AJ312437; Telosma mosaic virus DQ851493; Passionfruit woodiness virus HQ122652; Cowpea aphid-borne mosaic virus AF348210; Bean common mosaic necrosis virus HQ HQ229995, AY864314, AY282577, AY & U19287; Zucchini yellow mosaic virus L29569, AF014811, AJ515911, AB369279, AY AY279000, AM422386, AJ316228, AJ307036, L31350, AY188994, AJ & AB188115; Algerian watermelon mosaic virus EU B) same as in A, but trimmed to leave the sequence corresponding to the mature CP gene and including isolates AB (BCMV-IYbn), DQ (BCMV-VN/BB2-5) & DQ (BCMV-VN/YB2).

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