Molecular detection and characterisation of Horsegram Yellow Mosaic Virus (HgYMV) infecting Lima bean (Phaseolus lunatus) in India

The present study developed a PCR protocol using designed sequence virus-specific sets of primers (HYMV-A1500F & HYMV-A1500R and D-HYMV-B2200F & D-HYMV-B2200R) for the amplification of the complete DNA-A and DNA-B components of lima bean isolate of Horsegram yellow mosaic virus (HgYMV-Lb). The PCR products were cloned and sequenced. The nucleotide sequences of HgYMV-Lb was determined and compared with those of other whitefly-transmitted geminiviruses. The length of the single-stranded DNA-A and DNA-B components of HgYMV-Lb were 2735 and 2670 nucleotide, respectively. The DNA-A sequences were found most similar to the corresponding sequences of begomovirus species infecting legumes by up to 97% nucleotide identity. The DNA-B sequences were most similar to isolates of both Mungbean yellow mosaic virus and Mungbean yellow mosaic India virus at approximately 70% nucleotide identity. The genome organisation of HgYMV-Lb was also similar to that of a typical begomovirus bipartite genome. The DNA-A component has six Open Reading Frames (AV1/CP, AV2, AC1/REP, AC2/TrAP, AC3/REn, and AC4) and DNA-B has two (BC1 and BV2). The number, size and arrangement of the Open Reading Frames in the genome were similar to the Old world begomoviruses. In addition, phylogenetic analysis of DNA-A sequence clustered HgYMV-Lb into a group of legume-infecting begomoviruses from the Indian sub-continent, particularly with Mungbean yellow mosaic virus. Based on DNA-B sequences, HgYMV-Lb was clustered into the group with Mungbean yellow mosaic virus infecting soybean in Madurai (MYMV-Sb:Mad) and Mungbean yellow mosaic India virus in Bangladesh (MYMIV-BD:Yb). Thus, based on the PCR and sequence analysis, the association of a bipartite begomovirus with lima bean showing symptoms yellow mosaic disease in India was confirmed. Also, it was identified that the begomovirus was a novel isolate of previously described Horsegram yellow mosaic virus.


Introduction
. Sieve bean, butter bean, and baby lima bean Amongst the viruses, Yellow mosaic virus has varieties originate from second domestication been considered as an economically important because which occurred in Central America, probably in of its effect on the growth and yield of many legume Guatemala although the earliest archaeological species such as Cajanas cajan, Glycine max, hairy indigo evidence is from Mexico dated to 800 AD (Indigofera hirsute), Macrotyloma uniflorum, Phaseolus (Gobertson, 2004). However, Lima beans are now lunatus, Phaseolus vulgaris, Vigna mungo and Vigna grown all over the world and eaten as either cooked radiate (Muniyappa et al., 2008). It produces yellow or as fresh vegetable. mosaic symptoms and is easily transmitted by whitefly Lima beans are rich in fiber which helps to vector Bemisia tabaci (Order Hemiptera, family: eliminate cholesterol from the body (Gobertson, Aleyrodidae) (Pant et al., 2001;Maruthi et al., 2006;2004). The beans are a good source of potassium, Qazi et al., 2007). Initially, the symptoms caused by iron, copper and manganese. As it contain high Yellow mosaic virus appear as small yellow specks along potassium and low sodium, thus help reduce blood the veins and then spread over the leaf and are largely pressure (Kathirvel and Kumudha, 2011 (Collins, 2007; begomoviruses (Pant et al., 2001;Maruthi et al., 2007;Kathirvel and Kumudha, 2011).The wild Lima bean seeds have high levels of glucosides which Qazi et al., 2007). Thus, Yellow mosaic virus infecting lima bean from India sub-continent has not yet been breakdown to toxic hydrocyanic acid when the characterized. The present study developed PCR seeds are bruised or chewed. However, modern protocol for HgYMV which infects lima bean that domesticated varieties, particularly those with originated from the Indian sub-continent and white seeds have minimal quantities of hydrocyanic characterized its genome structure and genome acid and are not toxic. Cooking in boiling water also organization, and also established its genetic destroys the cyanogens (Gobertson, 2004 degenerate primer sets were cloned and sequenced and Horsegram yellow mosaic virus (HgYMV). These (Rojas et al., 1992;Maruthi et al., 2007). The sequences viruses cause yield losses to a number of important obtained were used to design sequence (virus) specific pulse crops including horsegram and lima bean in sets of primers for the amplification of the complete DNAthe tropical and sub-tropical regions. The disease A and DNA-B of the begomovirus. The sequences were incidences can be high, often ranging from 50-100 aligned using either MEGA5 software programme. The percentdepending on the cultivar susceptibility and primers, HYMV-A1500F & HYMV-A1500R and HYMV-B time of infection (Govindu, 1964;Fauquet et al., 2200F & D-HYMV-B2200R were designed to amplify full-2003, Maruthi et al., 2006). length DNA-A and DNA-B components, respectively for 1 hour and centrifuged at 12,000×g for 10 minutes at o ( Table 1). All the primers were used at annealing 4 C. The pellet was washed in 0.5 ml 70% ethanol, centrifuged for 5 minutes and vacuum-dried for 5 minutes temperatures ranging 52-55 C.
in a Spin Vac. The pellet was dissolved in 1xTE buffer and o stored at -20 C till use.

Extraction of DNA from virus infected leaves
Total DNA was extracted from leaf tissue Amplification of complete genome of HgYMV in infected collected from lima bean plant showing typical begomovirus leaf symptoms, using cetyltrimethyl leaves ammonium bromide (CTAB) procedure as described All the DNA samples were subjected to series of by Taylor and Powell (1982) and modified by PCRs, with primers that directly amplify specific regions Maruthi et al. (2002). About 100 mg of leaf tissue (AV1/CP, AV2, AC1/Rep, AC2/TrAP, AC3/REn, and AC4 was ground thoroughly in a thick-gauged plastic for DNA-A and BV1/NSP and BC1/MP for DNA-B) in the bag using a hand-held ball bearing sample grinder genome of the begomovirus. PCR was performed using (Bioreba AG, Reinach, Switzerland) and mixed Red hot polymerase kit (Thermo Fisher Scientific Ltd., using a wallpaper seam roller in 10 volumes (1 ml  sequenced in both directions using T7 and SP6 primers.

Cloning and sequencing of the complete genome of
The sequences were edited and aligned using the HgYMV software package MEGA5 (Tamura et al., 2007). BLAST The PCR products obtained from the search analysis was carried out to confirm the identity of amplification of the full-length DNA-A (2.8 kb) using the sequences. Maximum parsimony analysis and HYMV-A1500F & HYMV-A1500R and DNA-B (2.7 kb) heuristic search were used to generate the most components of HgYMV-Lb usingD-HYMV-B 2200F & parsimonious phylogenetic tree. The full-length DNA-A D-HYMV-B2200R were cloned into pGEMT Easy and DNA-B sequences of the two isolates from DNA-A vector (Promega, UK) and transformed to bacterial component and the three isolates from DNA-B were strains Escherichia coli (Promega, UK).The services compared with those reference Begomovirussequences of a commercial company (MRC Gene services Ltd, obtained from the National Center for Biotechnology Cambridge, UK) were used for plasmids DNA Information (NCBI) database. sequencing. For each sample two clones were  Total DNAs were extracted from leaves of nucleotides, respectively (Fig. 1). A CR was identified lima bean. The modified DNA extraction protocol in DNA-A and DNA-B sequences of the virus. The provided enough quantity and quality of DNAs common region (CR) contained the nona-nucleotide sufficient to carry out over 100 PCR reactions.

Total DNA extraction from virus infected leaves
sequence TAATATTAC that is conserved in the stem However, initially, there were some problems in loop of all begomoviruses, which has the DNA nicking amplifying viral DNA fragments. This may be due to site for the initiation of replication. The total numbers various reasons including the presence of impurities of open reading frames (ORFs) in begomoviruses and inhibitors in DNA samples. Therefore, the DNA predicted are identified for each virus component extraction protocol was modified by replacing (Fig. 2). DNA-A component was predicted to encode Phenol:Chloroform:Isoamyl alcohol (25:24:1) with six genes (AV1/CP, AV2, AC1/Rep, AC2/TrAP, Chloroform:Isoamyl alcohol (24:1), which produced AC3/REn, and AC4) and DNA-B component to encode DNAs with less impurities. two genes (BV1/NSP and BC1/MP) ( Table 2). The genes encoded for both the virion and complementary sense strands with their size and Genome organisation of HgYMV-Lb arrangements were typical of previously reported The full-length DNA-A and DNA-B component HgYMV and other begomoviruses. PCR products of HgYMV-Lb were 2735 and 2670

Genetic variability of HgYMV-Lb with other yellow mosaic viruses), begomoviruses infecting other hosts in Asia and Africa (Viruses infecting tomatoes, Begomoviruses
cassava, okra, soyabean, and cotton), in which HgYMV-Sequences obtained were identified Lb was grouped in HgYMV isolates. The branch tree of (closest homology sequence) using BLAST search.
HgYMV isolates showed less sequence variation among The DNA-A sequences were found most similar to these isolates. The branching out of different MYMIV the corresponding sequences of begomovirus and MYMV isolates suggested that these were entirely species infecting legumesby up to 97% nucleotide different viruses. Similar groupings were also observed identity. The DNA-B sequences were most similar to in phylogenetic tree obtained using DNA-B nucleotide isolates of both Mungbean yellow mosaic virus and sequences except that HgYMV-Lb clustered into group The From the results obtained, it was evident that the virus phylogenetic tree obtained using the DNA-A associated with yellow mosaic disease of lima bean is nucleotide sequences showed two clusters (Fig. 3), more closely related to other HgYMV isolates from begomoviruses infecting legumes in Asia different regions, rather than MYMIV and MYMV. (Mungbean yellow mosaic India viruses, Mungbean