Mungbean Yellow Mosaic India Virus Research Articles

Field and agroinoculation screening of national collection of urd bean (Vigna mungo) germplasm accessions for new sources of mung bean yellow mosaic virus (MYMV) resistance.

Yellow mosaic disease (YMD) is a major problem in Urd bean (Vigna mungo L.) in India, which causes huge yield losses. Breeding for wide spectrum and durable Mungbean yellow mosaic virus (MYMV) resistance and cultivating resistant cultivars is the most appropriate and effective approach. However, the task has become challenging with the report of at least two species of the virus, viz., Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV) and their recombinants; the existence of various isolates of these species with varied virulence and rapid mutations noted in the virus as well as in the whitefly vector population. Thus the present study was carried out to identify and characterize novel and diverse sources of YMV resistance and develop linked molecular markers for breeding durable and broadspectrum resistant urdbean cultivars against YMV. Towards this goal, we have screened 998 accessions of urdbean national collection of germplasm against YMD Hyderabad isolate both in a field under the natural level of disease incidence and through agro inoculation in the laboratory using viruliferous clones of the same isolate. Ten highly resistant accessions identified through repeated testing have been characterized in terms of reported linked markers. We attempted to see diversity among the ten resistant accessions reported here using earlier reported resistance-linked SCAR marker YMV1 and SSR CEDG180 marker. SCAR marker YMV1 did not amplify with any of the 10 accessions. But with CEDG180, results suggested that 10 accessions shortlisted through field and laboratory tests do not carry PU31 allele and this shows that it may be likely to carry novel gene(s). Further studies are needed to genetically characterize these new sources.

Deciphering the role of alternative splicing as modulators of defense response in the MYMIV- Vigna mungo pathosystem.

Alternative splicing (AS) is a crucial regulatory mechanism that impacts transcriptome and proteome complexity under stressful situations. Although its role in abiotic stresses is somewhat understood, our understanding of the mechanistic regulation of pre-mRNA splicing in plant-pathogen interaction is meagre. To comprehend this unexplored immune reprogramming mechanism, transcriptome profiles of Mungbean Yellow Mosaic India Virus (MYMIV)-resistant and susceptible Vigna mungo genotypes were analysed for AS genes that may underlie the resistance mechanism. Results revealed a repertoire of AS-isoforms accumulated during pathogenic infestation, with intron retention being the most common AS mechanism. Identification of 688 differential alternatively spliced (DAS) genes in the resistant host elucidates its robust antiviral response, whereas 322 DAS genes were identified in the susceptible host. Enrichment analyses confirmed DAS transcripts pertaining to stress, signalling, and immune system pathways have undergone maximal perturbations. Additionally, a strong regulation of the splicing factors has been observed both at transcriptional and post-transcriptional levels. qPCR validation of candidate DAS transcripts with induced expression upon MYMIV-infection demonstrated a competent immune response in the resistant background. The AS-impacted genes resulted either in partial/complete loss of functional domains or altered sensitivity to miRNA-mediated gene silencing. A complex regulatory module, miR7517-ATAF2, has been identified in an aberrantly spliced ATAF2 isoform that exposes an intronic miR7517 binding site, thereby suppressing the negative regulator to enhance defense reaction. The present study establishes AS as a non-canonical immune reprogramming mechanism that operates in parallel, thereby offering an alternative strategy for developing yellow mosaic-resistant V. mungo cultivars. This article is protected by copyright. All rights reserved.

Development of Specific Detection for Mungbean Yellow Mosaic India Virus Infecting Yard Long Bean in Java, Indonesia

Yellow mosaic disease was reported for the first time in Indonesia in 2008. Its infection on yard long bean caused significant yield loss. Mungbean yellow mosaic India virus (MYMIV), member of genus Begomovirus was identified as the main causal agent. Specific and accurate detection is important for disease monitoring as part of disease management strategy. The aim of this study was to construct specific primer pairs for quick and robust detection of MYMIV using polymerase chain reaction method. A pair of primers MY1/MY2 was designed in this study to amplify part of MYMIV coat protein. In silico and in vitro test showed that MY1/MY2 primers specifically amplified MYMIV.

Molecular Epidemiology of Begomoviruses Infecting Mungbean from Yellow Mosaic Disease Hotspot Regions of India.

The major threat to mungbean (Vigna radiata L.) cultivation in the Indian subcontinent is yellow mosaic diseases (YMD), caused by Begomovirus containing bipartite genomes (DNA-A and DNA-B). In the current study, we address the epidemiology of begomoviruses infecting mungbean plants in three YMD hotspot regions of India. Full-length genomic components of the viruses from the symptomatic leaves were cloned by rolling circle amplification (RCA) and sequenced. Mungbean yellow mosaic virus (MYMV) was detected in Bihar and mungbean yellow mosaic India virus (MYMIV) in Assam and Orissa. Furthermore, we studied the population structure and genetic diversity of MYMV and MYMIV isolates of Vigna species reported to date from India. Interestingly, based on phylogenetics, we observed independent evolution of DNA-A and coevolution of DNA-B of MYMV and MYMIV. This finding is supported by the high mutation rate and recombination events in DNA-B, particularly in BV1 and BC1 genes over DNA-A, with high transition/transversion bias (R) for DNA-A over DNA-B. To investigate the effect of Begomovirus infection in plants, we constructed infectious clones (i.e. MYMV and MYMIV) and inoculated them to eight mungbean genotypes, cowpea (Vigna unguiculata L.) and tobacco (Nicotiana benthamiana) through agroinfiltration. The infected plants developed varying degrees of typical YMD symptoms. Based on the disease severity score and viral titre, mungbean genotypes were categorized as highly susceptible to MYMV (ML267) and MYMIV (K851) and immune to MYMV (PDM139, SML668) and MYMIV (Pusa Vishal). Conclusively, our findings may help prevent an epidemic of YMD in Vigna species and develop mungbean genotypes resistant to YMD via breeding programs.

Genetics for seed traits and Mungbean yellow mosaic India virus reaction in urdbean (Vigna mungo L. Hepper)

AbstractUrdbean or blackgram is one of the important multi‐season pulse crops grown in India. Improved seed quality and resistance to Mungbean yellow mosaic India virus (MYMIV) are the important objectives of the urdbean breeding program. For this, knowledge of inheritance for seed quality traits and MYMIV resistance is essentially required. Therefore, an attempt has been made to understand the inheritance of seed traits like seed luster, seed coat color, mosaic on seed surface, and MYMIV reaction using intra‐ and inter‐specific bi‐parental populations. The inheritance results revealed that in DPU88‐31 × LBG‐685 population, the shining seed registered dominance over the dull trait, while the green seed coat color exhibited dominance over the brown seed coat color. However, the inter‐specific F2 population derived from IPU11‐02 × Pant M 6 deviated significantly from Mendelian ratios and supported severe segregation distortion for seed traits, while the mosaic on seed coat or mottling character studied in interspecific population demonstrated a dominant digenic inheritance pattern. In five inter‐ and intra‐specific F2 populations, MYMIV resistance demonstrated monogenic dominant inheritance, which was also validated in F3 generations. Furthermore, linkage analysis exhibited an association between seed luster and seed coat color, while no association was noticed between seed traits and MYMIV resistance. The normality tests revealed that seed width and 100 seeds weight were controlled by a few major genes, while other quantitative traits were governed by many genes with small additive effects. The skewness suggested that complementary gene interactions were present among genes controlling 100 seed weight in the intra‐specific cross, while duplicate gene interactions were involved in the inter‐specific cross. The identified monogenic traits and linked morphological marker after further enrichment of the linkage group could be used as an important tool in the regular breeding program.

Development of improved genotypes for extra early maturity, higher yield and Mungbean Yellow Mosaic India Virus (MYMIV) resistance in soybean (Glycine max)

Context Breeding for early maturity and higher yield is the principal objective in genetic improvement of Indian soybean. Yellow Mosaic Disease caused by Mungbean Yellow Mosaic India Virus (MYMIV) causes 80% yield loss in soybean. Aims This study aimed to develop early maturing, MYMIV resistant and high yielding soybean genotypes for enhancing soybean production and expanding the land area under cropping. Methods MYMIV resistance was introgressed from G. soja in to a widely adaptable cultivar JS 335 through a series of four generations of backcrosses and by evaluating derived progeny against MYMIV at a disease hot spot. Key results An extra-early maturing (71 days) genetic stock called NRC 252 was developed, which can be a potential gene donor in breeding for early maturing soybean varieties. Introgression lines YMV 1, YMV 2, YMV 11 and YMV 16 with MYMIV resistance and higher yield performance over recurrent parent and other check varieties were identified and characterised. Biplot analysis, assessing the main effect of genotype and the interaction of genotype with environment, revealed an ideal genotype with respect to 100-seed weight and grain yield that was also promising under sugarcane-soybean intercropping system in spring season. Conclusions Alleles from wild type soybean could improve yield attributing traits and MYMIV resistance in cultivated soybean. Improved genotypes such as YMV 1, YMV 2, YMV 11 and YMV 16 were found superior to the recurrent parent JS 335 as well as other check varieties. Implications The genotypes developed in the present study will help in reducing the damage caused by MYMIV disease and expansion of the area of soybean cultivation through intercropping with sugarcane.

Identification of begomoviruses associated with the insect vector Bemisia tabaci and various host plants on Java Island, Indonesia.

Begomoviruses are economically important plant viruses and are transmitted by Bemisia tabaci which is a complex of various cryptic species. However, it is uncertain whether most begomoviruses that infect host plants are transmitted by B. tabaci at a similar rate. We compared the begomovirus profiles that were detected in a total of 37 whitefly populations and 52 host plants on Java Island, Indonesia. Seven begomovirus species were detected in B. tabaci at different rates: pepper yellow leaf curl Indonesia virus (PepYLCIV, 56.8%), tomato yellow leaf curl Kanchanaburi virus (TYLCKaV, 46.0%), tomato leaf curl New Delhi virus (ToLCNDV, 21.6%), squash leaf curl China virus (SLCCNV, 21.6%), ageratum yellow vein China virus (AYVCNV, 2.7%), mungbean yellow mosaic India virus (MYMIV, 2.7%), and okra enation leaf curl virus (OELCuV, 2.7%). The begomoviruses were detected at different rates in three cryptic species of B. tabaci. In addition, six begomovirus species were detected in the various host plants at different rates: PepYLCIV (67.3%), TYLCKaV (53.9%), ToLCNDV (13.5%), MYMIV (11.5%), AYVCNV (3.9%), and Tomato yellow leaf curl Thailand virus (TYLCTHV) (1.9%). By comparing the virus presence between whiteflies and plants, five begomoviruses (AYVCNV, MYMIV, PepYLCIV, ToLCNDV, and TYLCKaV) were detected in both samples, but their sequence similarity was highly variable depending on the begomovirus themselves; TYLCKaV was highest (99.4%-100%) than any other viruses. Our study suggests B. tabaci acquire begomoviruses at different rates from plants. This study provides important information on the potential variation in the begomovirus transmission mechanism.

A large-effect QTL introgressed from ricebean imparts resistance to Mungbean yellow mosaic India virus in blackgram (Vigna mungo (L.) Hepper).

Here, we report identification of a large effect QTL conferring Mungbean yellow mosaic India virus resistance introgressed from ricebean in blackgram variety Mash114. The tightly linked KASP markers would assist in marker-assisted-transfer of this region into Vigna species infected by MYMIV. Until recently, precise location of genes and marker-assisted selection was long thought in legumes such as blackgram due to lack of dense molecular maps. However, advances in next-generation sequencing based on high-throughput genotyping technologies such as QTL-seq have revolutionized trait mapping in marker-orphan crops. Using QTL-seq approach, we have identified a large-effect QTL for resistance to Mungbean yellow mosaic India virus (MYMIV) in blackgram variety Mash114. MYMIV is devastating disease responsible for huge yield losses in blackgram, greengram and other legumes. Mash114 showed consistent and high level of resistance to MYMIV since last nine years. Whole genome re-sequencing of MYMIV-resistant and susceptible bulks derived from RILs of cross KUG253 X Mash114 identified a large-effect QTL (qMYMIV6.1.1) spanning 3.4Mb on chromosome 6 explaining 70% of total phenotypic variation. This region was further identified as an inter-specific introgression from ricebean. Linkage mapping using KASP markers developed from potent candidate genes involved in virus resistance identified the 500kb genomic region equaling 1.9cM on genetic map linked with MYMIV. The three KASP markers closely associated with MYMIV originated from serine threonine kinase, UBE2D2 and BAK1/BRI1-ASSOCIATED RECEPTOR KINASE genes. These KASPs can be used for marker-assisted transfer of introgressed segment into suitable backgrounds of Vigna species.

NMR-Based Metabolomic Profiling of Mungbean Infected with Mungbean Yellow Mosaic India Virus.

Mungbean is an important legume mainly cultivated in Southeast Asia known for cheap source of food protein. Yellow mosaic disease (YMD) of mungbean is one of the most damaging diseases caused by mungbean yellow mosaic virus (MYMV) and mungbean yellow mosaic India virus (MYMIV) in India. The genetic basis of YMD resistance of mungbean is not well studied yet. Our present studies aimed to explore the genetic basis of YMD resistance through molecular, biochemical and metabolomics approach. Molecular analysis of YMV-infected mungbean plant materials revealed the presence of MYMIV. Chlorophyll contents were estimated as mosaic symptoms that cause chlorosis and necrosis in infected leaves. Chlorophyll a, b and total chlorophyll content were significantly reduced by 27-55% in infected samples compared non-infected control samples. 1H NMR-based metabolomic profiling of virus-infected mungbean were carried out, and we found that vital changes occurred during the development of MYMIV infection in mungbean. A total of fifty metabolites were identified in mungbean leaf samples. Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) separated the severely infected sample from the non-infected samples. Orthogonal partial least discrimination analysis (OPLS-DA) revealed significant differences in MYMIV-infected and non-infected control samples. The featured metabolites in MYMIV infected and control samples were amino acids, carbohydrates, and organic acids. Relative abundance of sucrose, γ-amino butyric acid (GABA), proline, alanine, phenylalanine, tryptophan, pyruvate, ascorbate, and citrates were found as differential metabolites. Our results suggest that metabolic changes in infected mungbean samples is related to the viral acquisition. The present study may help in better understanding the metabolic alterations during biotic stress in mungbean.

First Report of Cowpea Polerovirus 2 and Southern Cowpea Mosaic Virus Infecting Cowpea in Nepal.

Cowpea (Vigna unguiculata L. Walp) is one of the important legume crops of Nepal, which is consumed as a green vegetable or a dried pulse. In recent years, virus diseases have caused significant yield and quality losses in cowpea. In September 2019, five cowpea plants showing virus-like symptoms of mosaic, yellow mosaic, vein clearing, chlorotic spots, (Fig. S1) were collected in Chitwan, Nepal. The incidence of symptomatic plants in the three kitchen gardens was about 10-20%. To identify the viruses associated with the disease, a pooled sample from all five plants was screened initially by next generation sequencing (NGS). Total RNA was extracted from the symptomatic leaves using RNeasy Plant Mini Kit (Qiagen, Germany) and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep kit (Illumina, San Diego, CA) according to the standard protocol. NGS was performed using an Illumina NovaSeq 6000 system (Macrogen Inc. Korea). A total of 324,807 contigs in the range of 201-14,645 nucleotides (nt) were obtained and analyzed against the viral reference genome database in GenBank by BLASTn and BLASTx search. Among the analyzed contigs, two large contigs showed homologies to cowpea polerovirus 2 (CPPV2) and southern cowpea mosaic virus (SCPMV. The CPPV2 contig (361,121 mapped reads, mean read coverage of 9,206.4 times) had a nearly complete genome sequence of 5,923 nt and showed 96% identity (99% coverage) with CPPV2 isolate BE179 (GenBank Acc. No. KY364847) isolaed from cowpea in Burkina Faso (Palanga et al. 2017). The SCPMV contig (10,612 mapped reads, mean read coverage of 384.1 times) had a nearly complete genome sequence of 4,172 nt and showed 90% identity (100% coverage) with SCPMV isolate C (GenBank Acc. No. M23021) isolated from cowpea in the USA (Wu et al. 1987). Additionally, mungbean yellow mosaic India virus (MYMIV, genus Begomovirus) and bean common mosaic virus (BCMV, genus Potyvirus) were detected at very low read depths. To confirm the presence of these viruses, total RNA was extracted from individual leaf samples, and reverse transcription PCR (RT-PCR) was performed using specific primers for each virus (Table S1). Three of five cowpea samples were positive for CPPV2, and they were co-infected with one other virus; SCPMV, MYMIV, or BCMV (Fig. S1). One cowpea sample was positive for the remaining one with symptom of overall chlorosis was negative for all four viruses. The amplified products of 1,205 bp for CPPV2 isolates, CPPV2-NP10, -NP12, and -NP24 were sequenced and deposited in GenBank under accession nos. MZ318692-93. These Sanger sequences shared 99% nt identity with the NGS-derived sequence. The amplified product of 1,394 bp for SCPMV isolate SCPMV-NP12 (GenBank acc. no. MZ355623) shared 100% nt identity with the NGS-derived sequence. CPPV2 is a member of the genus Polerovirus and it was first identified and characterized by molecular assays in Burkina Faso (Palanga et al. 2016; 2017). SCPMV is a member of the genus Sobemovirus and it has been reported in the USA, China, and Burkina Faso (Hull et al. 2000; Lee et al. 2001; Wu et al. 1987). In Nepal, MYMIV has been reported in legumes, such as kidney bean, black gram, and mungbean, and BCMV in common bean (Acharya and Regmi 2020). To our knowledge, this is the first report of CPPV2 and SCPMV in cowpea in Nepal. Further work is required to determine the distribution, pathological properties, and economic impact of these two viruses.

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

Mungbean yellow mosaic India virus (MYMIV) is a representative of the genus begomovirus/Begomoviridae, which is prevalent in the northern part of Indian subcontinent causing yellow mosaic disease (YMD). This virus is rapidly evolving and breaking the resistance in the advanced lines causing huge economic losses in the pulse production. In this context, the present investigation on characterization of the causal organism of YMD was undertaken METHODS AND RESULTS: A novel recombinant isolate (YMV-BG-BPT) causing YMD was identified from blackgram in Andhra Pradesh, southern peninsular region of India. The association of a bipartite begomovirus with the disease was done by sequence analyses of the cloned full-length genome. The full length genome sequences were submitted in NCBI GenBank with accession numbers MZ235792 (DNA-A) and MZ356197 (DNA-B). The sequence analysis of DNA-A of YMV-BG-BPT showed maximum of 99.12% similarity at nucleotide level with Mungbean yellow mosaic India virus (MYMIV) isolate reported from Tamil Nadu (KC911719), India which is also confirmed by clustering pattern in phylogenic analysis and DNA-B showed 95.79% with Mungbean yellow mosaic virus (MYMV) isolate reported from Tamil Nadu (KP319016) and 95.05% with MYMIV isolate reported from Karnataka (MT027037). The huge variation in DNA-B lead us to suspect a recombination in DNA-B, where a recombination event in the CR, region coding for nuclear shuttle protein and movement protein of DNA B was detected in which MYMV-BG-AP-IND (KF928962) and MYMIV-GG-CH-IND (MN020536) have been identified as major and minor parents, respectively. Overall, the present study revealed occurrence of MYMIV with recombinant DNA B component in southern peneinsular India.

Development of agro-infectious clones for screening resistance against recombinant mungbean yellow mosaic India virus causing golden mosaic disease in vegetable cowpea.

The online version contains supplementary material available at 10.1007/s13205-022-03206-2.

Biochemical alterations in soybean (Glycine max) infected with Mungbean yellow mosaic India virus in Lalitpur dist., Bundelkhand region of India

The yellow mosaic symptoms on soybean (Glycine max) were observed at various sites in the Bundelkhand region of India and infection of begomovirus was detected through PCR. The begomovirus isolates were identified as Mungbean yellow mosaic India virus (MYMIV). MYMIV infection in soybean results in the production of reactive oxygen species to activate defense mechanisms at different levels to overcome the damage caused in the severely infected crops. The biomarkers of stress in plants such as, hydrogen peroxide (H2O2), catalase (CAT), Superoxide dismutase (SOD), Ascorbate peroxidase (APX) contents studies also suggested elevation in stress-related enzymes and also a significant reduction in sugar, chlorophyll, and protein contents have been observed. Previously alteration in biochemical due biotic and abiotic stresses have been reported in different varieties of legumes, but biochemical alterations in soybean infected by MYMIV are being reported for the first time from the Bundelkhand region of India.

Species diversity, phylogeny and evidence of recombination in begomoviruses associated with yellow mosaic disease of moth bean (Vigna aconitifolia) in South India

AbstractPolymerase chain reaction assays of eight yellow mosaic disease (YMD) affected moth bean samples using specific primers indicated the presence of Mungbean yellow mosaic virus (MYMV), Mungbean yellow mosaic India virus (MYMIV) and Horsegram yellow mosaic virus (HgYMV) in two samples (Mo1 and Mo5) and two of these three viruses in remaining six samples. Sequence data analysis of Mo1 and Mo2 revealed the involvement of multiple DNA‐A and DNA‐B molecules in both samples. Further analysis of DNA‐A and DNA‐B molecules confirmed the presence of three viruses viz., MYMIV, MYMV and HgYMV, in Mo1 and of two MYMV and HgYMV in Mo2 samples. Analysis of common region (CR) sequences of components (DNA‐A and DNA‐B) of each virus indicated >85% nucleotide identity confirming them to be cognate and species integrity. The recombination analysis revealed the presence of a total of six putative recombination events in the viruses under study. In a phylogenetic analysis of DNA‐A sequences of the viruses under study with bipartite legume‐infecting begomoviruses and some selected old and new world begomoviruses, MYMIV, MYMV and HgYMV grouped with respective virus species. Similar grouping and clustering were found in the DNA‐B sequences also. The present study reports infection by HgYMV in moth bean and the association of three viruses (MYMIV, MYMV and HgYMV) with moth bean yellow mosaic disease for the first time. The presence of mixed infection of begomoviruses provides opportunities for the exchange of DNA between the species which may lead to the development of new strain/virus species.

New record of mungbean yellow mosaic India virus in Desmodium laxiflorum and association of bhendi yellow vein mosaic betasatellite in Abelmoschus moschatus in Andhra Pradesh, India.

Mungbean yellow mosaic India virus (MYMIV) is one of the most serious commonly occurring yellow mosaic virus (YMV's) group in majority of the pulses especially black gram and green gram in southern India compared to previously reported mungbean yellow mosaic virus. In January 2020 Desmodium laxiflorum and Abelmoscus moschatus showing mosaic symptoms and vein yellowing were collected from Guntur and Prakasam districts respectively in Andhra Pradesh. PCR analysis using MYMIV and betasatellite specific primers gave desired expected amplification from the infected samples of A. moschatus (YMV-ABEL) whereas only MYMIV specific amplification was obtained in D. laxiflorum (YMV-DES). However, no PCR amplification was obtained in respective healthy leaf samples of both plants. Sequence analysis showed that the CP sequence of YMV-ABEL and YMV-DES showed a similarity of 99.19% with MYMIV (KP677496) and 99.75% with MYMIV (JN181003) respectively. The full-length betasatellite (1356bp) showed highest identity of 90% with bhendi yellow vein mosaic betasatellite (BYVMB) (GU111977). Phylogenetic analysis clustered the test isolates with south Indian isolates of MYMIV whereas the betasatellite sequence clustered with various isolates of BYVMB, tomato leaf curl New Delhi virus betasatellite and okra leaf curl betasatellite reported from India and Pakistan. To the best of our knowledge, this is the first report of a MYMIV in D. laxiflorum and A. moschatus and MYMIV betasatellite complex in A. moschatus.

Construction of Full-length Dimer Clones of Yellow Mosaic Virus and Screening of Blackgram Germplasm using Agroinoculation

Background: Vigna mungo (Blackgram) is the major pulse crop cultivated in the Indian sub-continent. It is highly prone to yellow mosaic disease (YMD), which is widespread problem because all cultivated varieties are susceptible to the YMD in Andhra Pradesh state. The presence of two species of begomovirus i.e. Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV) with YMD was reported in Andhra Pradesh. The current study was aimed to develop simple method of viral dimer clone construction and identifying the resistant blackgram genotypes against two species of begomovirus (MYMV and MYMIV) by agroinoculation technique. Methods: In this study, we standardize the rolling circle amplification (RCA) based viral dimer clone construction (MYMV DNA-A, MYMV DNA-B and MYMIV DNA-A) and constructed viral dimer clones were used to transform Agrobacterium tumefaciens EHA105 cells through freeze-thaw technique. Sprouted seed method of agroinoculation screening was conducted. Result: Total 45 blackgram genotypes were screened by sprouted seed method of agroinoculation with two combinations of viral dimeric constructs (MYMV-TPT-A+MYMV-TPT-B and MYMIV-TPT-A+MYMV-TPT-B). Twenty four genotypes offered resistant against MYMV infection and twenty three genotypes were resistant to MYMIV infection. Total 17 genotypes offered resistance to both species of virus (MYMV andMYMIV) associated with YMD.

A Method for Developing RNAi-Derived Resistance in Cowpea Against Geminiviruses.

In plants, RNA interference (RNAi) is triggered by double-stranded RNA (dsRNA). Accordingly, various RNA silencing technologies involving hpRNA, artificial microRNA (miRNA), and virus-induced gene silencing (VIGS) are used for controlling the expression of genes. Such manipulations help understanding gene functions and crop improvement biotechnology. A typical hpRNA construct is comprised of an intron splicable perfect inverted repeat of the target gene sequences under the control of a strong promoter. Geminiviruses, especially Mungbean Yellow Mosaic India Virus (MYMIV) cause devastating diseases in legume plants including cowpea, incurring severe crop loss. RNAi, involving hpRNA construct as transgene, is used to control these diseases at the early stages of geminivirus infection in the host, preventing symptom development and viral DNA accumulation. In this chapter, we describe a detailed protocol for the identification of geminivirus isolates from the filed grown cowpea plants, characterization of virus isolates under the laboratory conditions, design and construct RNAi vectors for effective suppression of viral target genes, and consequent development of transgenic cowpea using Agrobacterium-mediated transformation protocol. These transgenics are subsequently evaluated for resistance to MYMIV.

Survey for Yellow Mosaic Disease Occurring on Blackgram [Vigna mungo (L.) Hepper] and Weed Hosts in Four Major Blackgram Growing Districts of Andhra Pradesh and Characterization of Associated Viruses using Coat Protein Gene

Background: Yellow mosaic disease (YMD) caused by Yellow mosaic virus is one of the major constraints in the pulse production in Andhra Pradesh (A.P.) due to fast evolution of strains, like Mungbean yellow mosaic India virus (MYMIV). Keeping this in view, a survey was undertaken in the major blackgram growing districts of A.P. to know the YMD incidence in blackgram and weed hosts and were characterized based on genetic features by comparing with other YMV isolates from different hosts and locations across the world. Methods: Roving survey was conducted during rabi 2019-20 in major blackgram growing districts of A.P. viz., Krishna, Guntur, West Godavari and Prakasam districts for YMD incidence. Blackgram plants showing characteristic symptoms were collected as representative samples from each mandal along with the suspected weed plants and were subjected to amplification using coat protein (CP) specific primers followed by molecular characterization. Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny. Result: Out of the four districts surveyed, the highest disease incidence was recorded at Machavaram village of Prakasam district (43.22%), whereas least disease incidence was recorded at Chinaganjam village of Praksam district (2.4%). Six weeds viz., Ageratum conyzoides, Amaranthus viridis, Parthenium hysterophporus, Vigna trilobata, Abelmoscus moschatus, Desmodium laxiflorum have showed positive result in PCR amplification with MYMIV specific coat protein primers. Four isolates from blackgram samples and two from weed plants shared 94.85 to 99.58% nucleotide identity among themselves.

Gamma-rays and EMS induced resistance to mungbean yellow mosaic India virus in mungbean [Vigna radiata (L.) R. Wilczek] and its validation using linked molecular markers

Purpose Mungbean yellow mosaic India virus (MYMIV) is a serious constraint in the mungbean which is a potential source of easily digestible high-quality proteins, fibers, minerals, and vitamins in Asian countries. Developing resistant cultivars is the most cost-effective, eco-friendly, and sustainable approach to protect mungbean from MYMIV damage. Mutation breeding provides a quick and cost-effective way of developing resistance as lack of genetic variability is the biggest bottleneck for other traditional breeding tools. Materials and methods Outstanding but MYMIV-sensitive varieties of mungbean, viz., MH 2-15 and MH 318 were mutagenized through various individual and combined doses of gamma-rays and Ethyl methanesulfonate (EMS) and evaluated in M2 and M3 generations for the appearance of resistance reactions. This was subsequently validated through marker-assisted genotyping using previously reported Yellow Mosaic Disease (YMD) linked markers. Results The phenotyping in M3 generation yielded 64 MYMIV resistant mutants whereas, marker-assisted genotyping identified the 22 mutants with true resistance. Markers YR4, CYR1, and CEDG180 were found associated with MYMIV resistance whereas, DMB-SSR158 did not show any amplification. Among identified resistant mutants, ten lines exhibited at par and two revealed a little higher seed yield over controls. Conclusions The mutagenesis created significant variability in MYMIV resistance as well as seed yield per plant. YR4, CYR1, and CEDG180 are found to be linked with the MYMIV loci in the mungbean and could be utilized for MYMIV resistance breeding. Mutant M-37 from MH 2-15 and M-104 from MH 318 exhibited more seed yield along with MYMIV resistance which upon further validation can be released as a variety. The induced mutagenesis integrated with powerful emerging molecular and next-generation sequencing (NGS) tools would be highly helpful in breeding mungbean for durable resistance against threatening MYMIV.

Resistance status of mungbean (Vigna radiata (L.) Wilczek) advanced breeding materials against mungbean yellow mosaic India virus

Seventy-one advanced breeding lines of mungbean were screened under epiphytotic conditions in the field for resistance against yellow mosaic disease (YMD) using infector technique during Kharif of 2016–2019 at Main Research Farm of IIPR, Kanpur. The identity of the YMD causing virus species was confirmed by PCR using specific primers and found to be as Mungbean yellow mosaic India virus. The average disease rating score recorded for test genotypes ranged from 1–4 as against 7.75 (1–9 disease rating scale) of susceptible check (DGGV-2). Of the 71 test genotypes, 48 showed a good level of resistance (1–2 scale) and of them, genotypes- IPM1205-2, IPM14-49-5, IPM14-10, IPM411-9, IPM709-5-2, IPM14-12-3, IPM14-2, IPM526-11 and IPM 08-11 consistently showed a high level of resistance against YMD for 3 or 4 years with a disease rating score of 1. These genotypes can be exploited as donors for YMD resistance.