Genetic diversity of legume yellow mosaic begomoviruses in Indonesia and Vietnam

High incidences of yellow mosaic symptoms were observed in soybean and yard‐long bean crops in Indonesia in 2009 and in mungbean crops in Vietnam in 2011. All five soybean and 20 yard‐long bean samples from Java, Indonesia, and 15 mungbean samples from Vietnam with symptoms tested positive for begomovirus infection by polymerase chain reaction (PCR) with primer pair PAL1v1978B/PAR1c715H. On the basis of collection location and the nucleotide sequence comparisons of the 1.5 kb begomoviral DNA‐A components amplified, a subset of samples comprising two soybean and six yard‐long bean isolates from Indonesia and five mungbean isolates from Vietnam were taken forward for more detailed examination. Sequence comparison and phylogenetic analysis of the full‐length sequences of all Indonesian and Vietnam isolates alongside other legume‐infecting begomoviruses revealed that all the isolates from Indonesia were Mungbean yellow mosaic India virus (MYMIV) strain‐A, and all from Vietnam were Mungbean yellow mosaic virus (MYMV) strain‐B. To the best of our knowledge, this is the first identification of MYMIV and MYMV associated with yellow mosaic of legumes in Indonesia and Vietnam, respectively. The epidemiological implications and potential consequences of the emergence of legume‐infecting begomoviruses on legume production in these areas of Southeast Asia are discussed.     

A survey of mixed Begomovirus infection in solanaceae and fabaceae at different altitudes in East Java,Indonesia

A multiplex primer set was developed to detect four Begomoviruses in East Java, Indonesia, i.e. Tomato leaf curl New Delhi virus (ToLCNDV), Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), Pepper yellow leaf curl Indonesia virus (PepYLCIV) and Mungbean yellow mosaic India virus (MYMIV). Survey at different altitudes found that begomoviruses infecting pepper, tomato and long bean were more variable, while in eggplant and string bean were more uniform. As a single virus, TYLCKaV infected eggplant, and sometimes tomato and pepper; PepYLCIV infected pepper, tomato and long bean; ToLCNDV only infected long bean and tomato at low frequency; and MYMIV infected beans. Mixed infection occurred more frequently in the low altitude areas. Subsequent examination indicated that Cucumber mosaic virus (CMV) and potyviruses were also responsible for diseased fabaceous. Our data suggest a relationship between altitudes and virus species occurrence. However, which viral species infects a crop is mainly influenced by the crop rather than by altitude.     

Genome-Wide SNP Identification and Characterization in Two Soybean Cultivars with Contrasting Mungbean Yellow Mosaic India Virus Disease Resistance Traits

Mungbean yellow mosaic India virus (MYMIV) is a bipartite Geminivirus, which causes severe yield loss in soybean (Glycine max). Considering this, the present study was conducted to develop large-scale genome-wide single nucleotide polymorphism (SNP) markers and identify potential markers linked with known disease resistance loci for their effective use in genomics-assisted breeding to impart durable MYMIV tolerance. The whole-genome re-sequencing of MYMIV resistant cultivar ‘UPSM-534’ and susceptible Indian cultivar ‘JS-335’ was performed to identify high-quality SNPs and InDels (insertion and deletions). Approximately 234 and 255 million of 100-bp paired-end reads were generated from UPSM-534 and JS-335, respectively, which provided ~98% coverage of reference soybean genome. A total of 3083987 SNPs (1559556 in UPSM-534 and 1524431 in JS-335) and 562858 InDels (281958 in UPSM-534 and 280900 in JS-335) were identified. Of these, 1514 SNPs were found to be present in 564 candidate disease resistance genes. Among these, 829 non-synonymous and 671 synonymous SNPs were detected in 266 and 286 defence-related genes, respectively. Noteworthy, a non-synonymous SNP (in chromosome 18, named 18-1861613) at the 149^th base-pair of LEUCINE-RICH REPEAT RECEPTOR-LIKE PROTEIN KINASE gene responsible for a G/C transversion [proline (CCC) to alanine(GCC)] was identified and validated in a set of 12 soybean cultivars. Taken together, the present study generated a large-scale genomic resource such as, SNPs and InDels at a genome-wide scale that will facilitate the dissection of various complex traits through construction of high-density linkage maps and fine mapping. In the present scenario, these markers can be effectively used to design high-density SNP arrays for their large-scale validation and high-throughput genotyping in diverse natural and mapping populations, which could accelerate genomics-assisted MYMIV disease resistance breeding in soybean.     

The DNA-1 like alphasatellites in association with the bipartite Mungbean yellow mosaic virus in natural infections

Yellow mosaic disease is the major limitation in the production of grain legumes in India. This disease is caused by bipartite begomovirus, Mungbean yellow mosaic virus. In addition to the bipartite genomic components, the yellow mosaic disease affected urdbean plants which contain satellite like DNA-1 component called as alphasatellites. The present study has been attempted to characterise the alphasatellites associated with Mungbean yellow mosaic virus. Nucleotide sequence analysis of alphasatellites showed 98% identity with Vernonia yellow vein Fijian alphasatellite, VYVFA (JF733780). Since the sequence identity is more than 98%, the threshold value for demarcation of alphasatellites species, the alphasatellites of the present study are named as Vernonia yellow vein Fijian alphasatellite. Comparison with other, alphasatellites shared 51–55% identity with alphasatellites associated with monopartite begomovirus and it shared only 41–42% identity with an unusual alphasatellites, DNA-2. This is the first report on characterisation of alphasatellites associated with Mungbean yellow mosaic virus.     

Characterisation of full genome of Dolichos yellow mosaic virus based on sequence comparison,genetic recombination and phylogenetic relationship

Yellow mosaic disease (YMD) is one of the most important diseases affecting different leguminous crops and causes significant yield losses in Indian sub‐continent. Eight different bipartite begomovirus species are known to cause YMD in more than 10 leguminous crops. These species are collectively known as legume yellow mosaic viruses (LYMVs), and their full genomes have been characterised except for Dolichos yellow mosaic virus (DoYMV). In this study, full genome of DoYMV isolate (KJ481204 and KJ481205) infecting dolichos has been characterised. The DNA‐A of DoYMV consists of 2761 nucleotides and DNA‐B of 2733 nucleotides with a genome organisation typical of Old World bipartite begomoviruses. Nucleotide identity of DNA‐B (KJ481205) of DoYMV with DNA‐B of other legumoviruses was 57.5–61.0%. Both components contain a nonanucleotide and conserved inverted repeat sequences with the potential to form a stem‐loop. Nucleotide identity of common region of DoYMV was 90.3%, above the threshold nucleotide identity (>85%) for considering a DNA‐B molecule as cognate of DNA‐A of a begomovirus. Four recombination events in DNA‐A and two in DNA‐B of DoYMV isolate were detected. Mungbean yellow mosaic virus, Rhynchosia yellow mosaic virus and Horsegram yellow mosaic virus were identified as probable parents.     

The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).     

Diagnosis of Symptomless Yellow mosaic begomovirus Infection in Pigeonpea by Using Cloned Mungbean yellow mosaic India virus as Probe

One isolate of Mungbean yellow mosaic India virus (MYMIV) of mungbean plants from Sri Ganganagar, Rajasthan, designated as MYMIV-Mg was isolated and DNA-A and DNA-B, the two full length bipartite genomic components of this virus, were cloned. The [α-³²P] labeled diagnostic probes specific to these cloned DNA-A and -B of MYMIV-Mg were used to detect the virus infection in infected plants by nucleic acid spot hybridization (NASH) test. The NASH tests detected the MYMIV infection and concentration of viral titre in susceptible, moderately susceptible, resistant and symptomless genotypes of pigeonpea (Cajanus cajan) plants. Fourteen genotypes of pigeonpea were tested against five naturally occurring MYMIV variants viz.,.MYMIV Bg, -MgD, -MoL, -Mg and -Pp1 through viruliferous whitefly (Bemisia tabaci) transmission in greenhouse condition. Disease incidence and severity of MYMIV in different pigeonpea genotypes varied with the variants of MYMIV. Many genotypes of pigeonpea did not produce visible yellow mosaic symptoms after inoculation with MYMIV variants MYMIV-Bg, -MbD and -MoL, although, majority of the symptomless genotypes were found to be infected by MYMIV, as viral DNA was detected by NASH test.     

Identification of Mungbean yellow mosaic Indian virus Associated with Tomato Leaf Curl Betasatellite Infecting Phaseolus vulgaris in Oman

Kidney bean (Phaseolus vulgaris) plants exhibiting foliar yellow mosaic symptoms and some leaf crumpling were identified in the Al Batinah region of Oman. Rolling circle amplification and polymerase chain reaction identified a bipartite begomovirus (family Geminiviridae) and a betasatellite in association with the symptomatic plants. Analysis of full‐length sequences showed the virus to be Mungbean yellow mosaic Indian virus (MYMIV) and the betasatellite Tomato leaf curl betasatellite (ToLCB). This is the first identification of a legume‐adapted begomovirus in Oman and the first identification of MYMIV in association with the betasatellite ToLCB. The isolate of MYMIV from Oman shows the greatest levels of sequence identity to isolates occurring in South Asia and South‐East Asia, suggesting that the virus has only recently been introduced. The significance of these findings is discussed.     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Molecular Marker-Assisted Genotyping of Mungbean Yellow Mosaic India Virus Resistant Germplasms of Mungbean and Urdbean

Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus begomovirus causes the yellow mosaic disease in a number of economically important edible grain legumes including mungbean (Vigna radiata), urdbean (Vigna mungo) and soybean (Glycine max). The disease is severe, critical, open spread and inflicts heavy yield losses annually. The objective of this study is to develop molecular markers linked to MYMIV-resistance to facilitate genotyping of urdbean and mungbean germplasms for MYMIV-reaction. Resistance-linked molecular markers were successfully developed from consensus motifs of other resistance (R) gene or R gene homologue sequences. Applying linked marker-assisted genotyping, plant breeders can carry out repeated genotyping throughout the growing season in absence of any disease incidence. Two MYMIV-resistance marker loci, YR4 and CYR1, were identified and of these two CYR1 is completely linked with MYMIV-resistant germplasms and co-segregating with MYMIV-resistant F₂, F₃ progenies of urdbean. The present study demonstrated that these two markers could be efficiently employed together in a multiplex-PCR-reaction for genotyping both V. mungo and V. radiata germplasms from field grown plants and also directly from the seed stock. This method of genotyping would save time and labour during the introgression of MYMIV-resistance through molecular breeding, as methods of phenotyping against begomoviruses are tedious, labour and time intensive.     

Characterization of Mungbean yellow mosaic India virus genome with a recombinant DNA-B in Southern Peninsular India.

Molecular Biology Reports - Mungbean yellow mosaic India virus (MYMIV) is a representative of the genus begomovirus/Begomoviridae, which is prevalent in the northern part of Indian subcontinent...     

Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.     

Molecular and biochemical characterization of mungbean yellow mosaic India virus resistance in leguminous host <Emphasis Type="Italic">Vigna mungo</Emphasis>

Mungbean yellow mosaic India virus (MYMIV)—the causal agent of the yellow mosaic disease is responsible for severe damage of crops that are of great economic importance. In the current study, we explored the process of MYMIV infection and its natural resistance by analysing the expression of early and late viral genes at different time points in the leaves of resistant and susceptible Vigna mungo plants. Accordingly, we have periodically evaluated several biochemical parameters commonly associated with oxidative status of resistant and susceptible V. mungo plants during MYMIV infection. Our study revealed that accumulation levels of the early as well as late expressed genes of MYMIV were low and high in the resistant and susceptible plants, respectively; whereas membrane stability index (MSI) exhibited an opposite response. Moreover, a decrease in the malondialdehyde levels along with an increase in the activities/levels of different antioxidant enzymes, total phenol and H₂O₂ was noted during the early stages of infection in the resistant plants. Such observations argue in favour of strong defensive capability of the resistant plants in restricting the accumulation of viral RNA and generation of harmful free radicals within the studied tissue. Collectively, it appears that obstruction of viral invasion in plant cell wall, restriction in viral DNA replication, and early onset of antioxidant defense responses altogether might be responsible for MYMIV natural resistance. Such information is helpful in understanding the pathogenesis of MYMIV infection and its resistance in V. mungo and other economically important crops.     

Comparative characterization of small RNAs derived from an emaravirus and a geminivirus infecting pigeonpea

High throughput sequencing technologies, supported by bioinformatics tools are employed to retrieve small RNA sequence information derived from the nucleic acids of plant infecting viruses. In addition to characterization of the small RNAs to understand the biology of the virus, the small RNA sequence can be assembled to reconstitute viral genome sequence. For the first time the semiconductor based Ion Proton sequencing technology is used to sequence the small RNAs from pigeonpea (Cajanus cajan) plants infected by two distinct viruses with RNA and DNA as their genomes. The reconstitution of the viral genome sequence revealed that the pigeonpea plant from Kalaburagi (erstwhile Gulbarga, Karnataka state) was infected by an emaravirus species Pigeonpea sterility mosaic emaravirus 1 (PPSMV-1) and another plant from New Delhi was infected by a begomovirus species Mungbean yellow mosaic India virus (MYMIV). Characterization and comparison of small RNA sequences derived from both the viruses showed vast differences in their pattern of accumulation and their size classes. In the case of PPSMV-1, the 21 nt sized siRNAs accumulated at far greater levels followed by 22 and 24 nt siRNAs. Whereas in MYMIV, the proportion of accumulation of each size class of siRNAs was similar. Further the distribution of small RNAs across the genomes of PPSMV-1 and MYMIV was mapped and the density of small RNA accumulation showed a positive correlation with the GC content of viral sequence.     

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem–loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.