Susceptible Mungbean Research Articles

Development of infectious clones of mungbean yellow mosaic India virus (MYMIV, Begomovirus vignaradiataindiaense) infecting mungbean [Vigna radiata (L.) R. Wilczek] and evaluation of a RIL population for MYMIV resistance.

Yellow mosaic disease (YMD) is a major constraint for the low productivity of mungbean, mainly in South Asia. Addressing this issue requires a comprehensive approach, integrating field and challenge inoculation evaluations to identify effective solutions. In this study, an infectious clone of Begomovirus vignaradiataindiaense (MYMIV) was developed to obtain a pure culture of the virus and to confirm resistance in mungbean plants exhibiting resistance under natural field conditions. The infectivity and efficiency of three Agrobacterium tumefaciens strains (EHA105, LBA4404, and GV3101) were evaluated using the susceptible mungbean genotype PS16. Additionally, a recombinant inbred line (RIL) population comprising 175 lines derived from Pusa Baisakhi (MYMIV susceptible) and PMR-1 (MYMIV resistant) cross was developed and assessed for YMD response. Among the tested Agrobacterium tumefaciens strains, EHA105 exhibited the highest infectivity (84.7%), followed by LBA4404 (54.7%) and GV3101 (9.80%). Field resistance was evaluated using the coefficient of infection (CI) and area under disease progress curve (AUDPC), identifying seven RILs with consistent resistant reactions (CI≤9) and low AUDPC (≤190). Upon challenge inoculation, six RILs exhibited resistance, while RIL92 displayed a resistance response, with infection occurring in less than 10% of plants after 24 to 29 days post inoculation (dpi). Despite some plants remaining asymptomatic, MYMIV presence was confirmed through specific PCR amplification of the MYMIV coat protein (AV1) gene. Quantitative PCR revealed a very low relative viral load (0.1-5.1% relative fold change) in asymptomatic RILs and the MYMIV resistant parent (PMR1) compared to the susceptible parent (Pusa Baisakhi). These findings highlight the potential utility of the developed infectious clone and the identified MYMIV-resistant RILs in future mungbean breeding programs aimed at cultivating MYMIV-resistant varieties.

Identification of important morphology for waterlogging tolerance from developed mung bean F2 population

Waterlogging is severe abiotic stress during mung bean (Vigna radiata (L.) R. Wilczek var. radiata) production. Waterlogging impacts yield loss for the legume crop by around 40%-70%, depending on the severity and duration of the waterlogging. In total, 107 collected mung bean varieties were evaluated under managed waterlogging. The F2 population from crossing between waterlogging-tolerant and -susceptible mung bean varieties were screened under waterlogging conditions. Morphological responses to the waterlogging were recorded. Correlation and path coefficient analyses were used to identify essential traits for waterlogging tolerance and seed yield potential under water stress. Mung bean varieties in clusters 5 and 6 were high waterlogging tolerance with survival rates of 66.66% and 61.11%, respectively. Of 199 F2 lines from crossings between the Kamphaeng Sean2 and W162, 20 lines presented high waterlogging tolerance. The injury score was negatively correlated with the number of leaves at the final date of waterlogging (-0.845) and 1 wk after terminating the waterlogging (-0.885). At the final date of waterlogging condition, the number of leaves and leaf greenness stability index directly affected leaf injury at -0.5928 and -0.4385. One week after waterlogging termination, only the number of leaves affected the leaf injury at -0.8599. The number of leaves directly affected seed yield components at 0.6534 and higher than the direct effect of the other traits. Thus, the number of leaves was a suitable morphological parameter along with the injury score for selecting waterlogging tolerance. The number of leaves was a suitable parameter for selecting high seed yield potential under waterlogging conditions.

Growth Performance and Metabolic Changes in Susceptible Mung Bean [Vigna radiata (L.) Wilczek] during Interaction with Rhizoctonia solani and Trichoderma virens

Background: Mung bean is susceptible to Rhizoctonia solani infection. Applications of beneficial microorganisms such as Trichoderma are promising for controlling pathogens and promoting plant growth. Methods: This study investigated growth performance and metabolic changes in mung bean seedlings during interaction with R. solani and Trichoderma virens using Gas Chromatography-Mass Spectrometry (GC-MS). Result: Mung bean infected by R. solani caused root rot and wilting. T. virens treatment reduced the disease severity in infected seedlings and promoted mung bean growth. Seventy-eight metabolites were identified in root extracts and dominated by sugars and fatty acids. The sugars, fatty acids and organic acids were significant metabolite groups that changed in response to pathogen infection and/or T. virens treatment. Five metabolic pathways particularly pyruvate metabolism, glyoxylate and dicarboxylate metabolism, sulfur metabolism, citrate cycle (TCA cycle) and phenylalanine, tyrosine and tryptophan biosynthesis altered significantly based on a metabolic pathway analysis. Acetic acid and aconitine had important roles in mung bean response to R. solani infection and/or T. virens treatment.

Bruchid Resistance Study through Bulked Segregant Analysis: Used as a Preliminary Step for Next-Generation Sequencing

Seed beetles, commonly known as bruchids, are serious insect pests of Indian pulses inflicting high damage during storage. Earlier, a bruchid resistant rice bean landrace (TNAU Red) from Manipur and a susceptible mung bean variety (VRMGg1) from Tamil Nadu were reported and their inter-specific cross was made in Tamil Nadu Agricultural University. In the present study, seeds from the segregating populations (recombinant inbred lines, RILs) of VRM (Gg)1 and TNAU Red were evaluated for bruchid Callosobruchus maculatus Fabricius resistance following standard methods under laboratory conditions. Bulked segregant analysis (BSA) is an efficient method for quick identification of molecular markers linked to any specific gene or genomic region. BSA significantly reduces the scale and cost by simplifying the procedure compared to conventional method of analyzing the entire population. BSA was carried out using 3 marker systems with an attempt to identify markers associated with the present trait of interest. Two DNA bulks namely resistant bulk (RB) and susceptible bulk (SB) were grouped by pooling equal amount of DNA from 5 each of resistant and susceptible RILs based on bruchid responses such as seed damage (%), mean developmental days (MDP) and adult emergence from seeds. Marker analysis carried out with 41 polymorphic SSRs showed skewed segregation distortion towards the susceptible parent. However, out of 9 RAPDs and 10 ISSRs, 2 RAPD (OPB08 and OPX04) and 1 ISSR (UBC810) primers amplified an allele at 200 - 300 and 300 - 400 bp, respectively, in resistant parent and resistant bulk but not in susceptible parent and susceptible bulk. The amplification of resistant parent allele in resistant bulk may indicate the presence of bruchid resistant gene(s) in the resistant RILs. This experiment was aimed at verifying the presence of bruchid resistant lines carrying resistant gene(s) prior to further association mapping or QTL mapping studies. Further, such type of study can be applied as a preliminary step in NGS technology.
 HIGHLIGHTS
 
 Bruchids are the most damaging storage insect in pulses and other legumes causing great economic losses
 Bulk segregant analysis (BSA) conducted using three marker systems in an attempt to identify markers associated with bruchid resistance
 Categorization of RILs based on bruchid responses such as seed damage (%), mean developmental days (MDP), adult emergence from seeds and scoring done
 Marker analysis using BSA useful for verification of the presence of bruchid resistant lines carrying resistant gene(s) prior to further advanced studies such as QTL mapping, NGS, etc.
 
 GRAPHICAL ABSTRACT

Identification of differentially expressed mungbean miRNAs and their targets in response to drought stress by small RNA deep sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs, 18–25 nt in length, that play a crucial role in regulating genes associated with the physiological processes and responses to various biotic and abiotic stresses. Different conserved and species-specific microRNAs and their functions have been identified, primarily in plants such as rice and Arabidopsis with sequenced genomes. Our present study identifies drought-responsive miRNAs and their potential targets from mungbean under three days of drought stress induced by PEG-6000. We constructed small RNA libraries from both control and drought-treated tolerant and susceptible mungbean genotypes and identified various miRNAs involved in drought stress regulation. Analysis of differentially expressed genes (DEGs) revealed 79 up-regulated and 158 down-regulated novel miRNAs and two up-and down-regulated known miRNAs under drought. Annotation of the miRNAs followed by target prediction and expression analysis revealed five miRNAs, Vra-miR160, Vra-miR164, Vra-miR167, Vra-miR394, and Vra-miR398, were potentially involved in the regulation of drought-responsive genes. Their predicted target genes were an Auxin response factor (ARF), a NAC (for petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) transcription factor, Serine acetyltransferase 1 , and Multicopper oxidase LPR2-like . The expression of drought-responsive miRNAs and their targets were validated by real-time PCR. Our data suggest that various known and novel microRNAs activated transcription factors, enzyme kinases, and hormone signaling pathways alleviate drought stress in the tolerant K-851 genotype of mungbean. • Our study identifies 79 novel up-regulated and 158 down-regulated miRNAs and their potential targets from mungbean under three days PEG induced drought stress using deep RNA sequencing. • The gene annotation of miRNAs followed by target prediction and their expression analysis revealed five miRNAs are likely to regulate drought-responsive genes, including various transcription factors, Serine acetyl transferases, and Multicopper oxidase LPR2-like genes. • This study shows that the expression of various novel microRNAs differs between the mungbean cv. K-851 (drought tolerant) and cv. PDM-139 (drought-sensitive) genotypes.

New Report of Cercospora canescens Isolates from Coastal Regions of Odisha, India causing Cercospora Leaf Spot (CLS) Disease in Mung Bean (Vigna radiata L.)

Background: Cercospora leaf spot (CLS) is one of the major diseases causing a major loss in production of mung bean of up to 60%. Although it is cultivated all round the world, its major contribution is attributed to the Asian continent especially the countries of Indian subcontinents which accounts for about 90% of the world’s mungbean production. Due to its high nutritional content, mung bean is a major contributor to the Nation’s economy. The present study was to isolate and characterize the new isolates of Cercospora canescens collected from coastal belts of Odisha, India. Methods: Surveys were conducted in the mung bean growing fields of Bhubaneswar (20.26°N, 85.8°E) and Berhampur (19.36°N, 84.77°E), coastal regions of Odisha during 2018-20. New Cercospora canescens isolates were collected and characterised through morphological, biochemical and molecular basis. Result: New Cercospora isolates were characterized on the basis of morphological, biochemical and molecular analysis to understand the gravity of the disease. Pathogenicity tests were conducted by artificial inoculation to identify the resistant and susceptible mung bean genotypes. On the basis of molecular analysis (internal transcribed spacer (ITS) region), there was a difference between the two isolates and forming different clades. These isolates were sequenced having the NCBI Acc. No. MZ475049 and MZ475050. Both isolates are different from each other because of transitional substitution of thymine and cytosine. The findings will be a complementary contribution for conservation and better management strategies of CLS disease in mung bean.

Identification of mung bean germplasm for resistance against Cercospora canescens and to study the association of biochemical parameters with defense mechanisms

AbstractSizable loss in crop yields resulting from Cercospora leaf spot (CLS), caused by Cercospora canescens and is a major disease in mung bean [Vigna radiata (L.) R. Wilczek] crops, demands a thorough evaluation of various parameters dealing with the infection. Determination of components for CLS resistance is associated with leaf spot intensity in the field, which is estimated from the area under the disease progress curve (AUDPC). The present study showed that out of 261 genotypes screened in the field for CLS, 88, 89, 51 and, 33 genotypes were found to be resistant, moderately resistant, moderately susceptible, and susceptible, respectively, on the basis of the AUDPC values. Further, five resistant and five susceptible genotypes were selected from the screened genotypes for bioassay analysis and were subjected to artificial inoculation with three different isolates of Cercospora canescens to study the effectiveness in polyhouse. The morphological data along with disease incidence, chlorophyll content, proline content, phenol content and enzymatic activities of phenylalanine ammonia‐lyase (PAL), peroxidase (POX), and superoxide dismutase (SOD) were estimated. The findings showed that there was a significant increase in the enzymatic activities, total phenol, and proline contents in inoculated resistant and susceptible genotypes vs. control (without inoculation). However, the total chlorophyll content reduced in both resistant and susceptible genotypes after inoculation. Moreover, higher enzyme activities were marked in the resistant genotypes than the susceptible genotypes. This study will help to identify the resistant and susceptible mung bean genotypes against Cercospora canescens for further agronomic study as well as breeding programs.

Inheritance of resistance to Mungbean Yellow Mosaic Virus (MYMV) in mungbean [Vigna radiata L. Wilczek

Six basic populations (P1, P2, F1, F2, B1 and B2) of 12 crosses involving two resistant and six susceptible mungbean genotypes were screened to study the inheritance pattern of MYMV resistance. All the 12 F1s were susceptible indicating that the susceptibility is dominant over resistance. The F2’s of each of the 12 crosses exhibited a segregation ratio of 9S: 3MS: 3MR:1R indicating the involvement of two recessive genes (r1 and r2) in the inheritance of MYMV resistance. When both genes were present together in a homozygous dominant state, R1- R2, plants were highly susceptible (S). In contrast, when both the genes were in homozygous recessive condition, r1r1,r2r2, the resistant reaction was obvious. However, when one gene was in homozygous recessive (R1,r2r 2) condition, moderate susceptibility (MS) was observed while, when the other gene was in homozygous recessive (r1r1, R2), condition, moderate resistant (MR) reaction was observed. Thus, the F2 segregation, 9 (R1R2): 3 (R1r2r 2): 3 (r1r1R2-): 1 (r1r1r2r 2), was explained on the basis of phenotypic expression, 9S: 3MS: 3MR:1R, which was further confirmed in a back cross generation. Key words: Mungbean, Inheritance, MYMV, Resistance

Screening of Mungbean [Vigna radiata (L.) Wilczek] Genotypes against Bruchid (Callosobruchus maculatus) Attack to Reduce Postharvest Losses

Background: Mungbean is an important pulse crop due to its high protein content. Bruchid is a major storage pest which causes deterioration of seed quality as well as quantity. The grain losses directly affect the agricultural economy and also the human health. Therefore, this issue has to be addressed with high priority. Methods: To address this issue, we have initiated a study to screen the fifty-two mungbean genotypes against the bruchid attack. The initial phase of the experiment was carried out from 2015 to 2017 in the Department of Genetics and Plant Breeding, University of Calcutta to the laboratory condition through the no choice test. Result: Using the parameter bruchid susceptibility index according to Dobie, four resistant, ten susceptible, seventeen moderately resistant and twenty-one moderately susceptible mungbean genotypes were identified. The host resistance genotype HUM-8, PM-11-51, APDM-84, VC-639 are identified as the most promising resistant genotype.

Exogenous Application of Elicitors Induces Biochemical Alterations and Develops Tolerance against Yellow Mosaic Disease in Mungbean

Mungbean (Vigna radiata) is seriously affected by mungbean yellow mosaic disease (MYMD) caused by Mungbean yellow mosaic India virus (MYMIV) in the Indian subcontinent. The present study was conducted to evaluate the effectiveness of salicylic acid (SA) and benzothiadiazole (BTH) for the management of MYMD and to find their role in inducing alteration in different biochemical parameters in susceptible mungbean genotype VC3061A. Exogenous application of the elicitors resulted in delayed symptom development and reduced disease severity (DS). The severity index (SI) was higher on positive control plants while the minimum was observed in post combined application of BTH+SA, followed by BTH and SA application. Total phenolic contents (TPC) and malondialdehyde (MDA) increased significantly in virus inoculated plants of all treatments as compared to their healthy controls. SOD activity was increased significantly in BTH+SA treated plants but decreased in disease control and BTH treated virus inoculated plants. A significant decrease in catalase (CAT) activity, while an increase in peroxidase (POD) was observed in BTH+SA, treated virus inoculated plants. Protease and esterase activity were significantly increased in SA treated virus inoculated plants. Plant pigments exhibited decreased concentration in virus inoculated plants compared to non-inoculated plants under all the treatments except SA treated plants. Enhanced or suppressed levels of antioxidants suggest an association between constitutive induced levels of these enzymes. In this study, we also report for the first time the protein profiling of mungbean genotype VC3061A after the exogenous application of different combinations of elicitors. Proteomic analyses revealed the expression of two proteins phosphatase 2C 16-like isoform and capsid protein after MYMIV inoculation in SA and BTH+SA treated plants which may trigger signal transduction pathway and consequently induces resistance against MYMIV in V. radiata by activating PR protein.

Biochemical Alterations in the Leaves of Resistant and Susceptible Mungbean Genotypes Infected with Mungbean Yellow Mosaic India Virus

Mungbean (Vigna radiata), an important leguminous crop, is highly susceptible to yellow mosaic disease (YMD) caused by Mungbean yellow mosaic India virus (MYMIV), resulting in high yield penalty. Previously, different varieties of mungbean have been reported possessing resistance/ tolerance against MYMIV. However, the molecular events occurring during compatible and incompatible interactions between mungbean and MYMIV are yet to be explored. Therefore, in this study using MYMIV-resistant (NM-2016), moderately resistant (NM-2011) and susceptible genotype (VC-1647C), alterations in various biochemical attributes due to MYMIV infection were analysed and compared with healthy non-inoculated control plants for understanding the resistance mechanism. After MYMIV inoculation, the level of total phenolic contents (TPC) and total soluble proteins (TSP) increased significantly in the susceptible genotype. However, the level of Malondialdehyde (MDA) and Ascorbate peroxidase (APX) remained same in all the genotypes. Level of superoxide dismutase (SOD) and catalase (CAT) decreased in the susceptible genotype but CAT level increased in the moderately resistant genotype. Protease level decreased significantly in all the genotypes while esterase level increased in moderately resistant and susceptible genotype. Peroxidase (POD) increased only in moderately resistant genotype and Total Oxidant Status (TOS) increased significantly in the susceptible genotype. Due to MYMIV infection the level of all plant pigments decreased in all the genotypes tested. The comparative proteome analysis using SDS-PAGE resolved 22 peptides with molecular weight from 12.5 to 163 kDa. Differential expression of protein phosphatase 2C (PP2C) and Cytochrome b6 (Photosynthesis) in resistant and moderately resistant genotypes; Pectin acetyl esterase and Resistant specific protein-1(4) in resistant genotype and up-regulation of superoxide dismutase [Cu-Zn] and RuBisco by MYMIV may have triggered signal transduction pathway and consequently induced a resistance response against MYMIV in V. radiata by activating PR proteins.

Evaluation of salt tolerant mungbean (Vigna radiata L.) Genotypes on growth through bio-molecular approaches

This study was conducted to obtain saline tolerant mungbean genotypes through evaluating growth, biochemical and molecular parameters, and possible salt tolerant mechanisms were studied in different salt sensitive genotypes. Thirteen prescreened mungbean genotypes were grown on 0, 40, 80 and 120 mMNaCl induced salinity and evaluated by germination percentage, shoot and root length, superoxide (O2•-) generation rate, concentration of H2O2, lipid peroxidation (as malondialdehyde, MDA), methylglyoxal (MG), K+/Na+ and proline content in leaves. Based on these parameters, genotypes BD-10588, BD-6894 and IR-01 were selected as tolerant genotypes. For studying oxidative stress tolerance mechanism, BD-10588 and IR-01 were used as tolerant and BD-6887 and BD-10741 as susceptible genotypes, and comparative ROS (O2•- and H2O2), and MDA as well as LOX activity between the two groups were determined. Analysis of activities of ROS metabolizing antioxidant enzymes strongly suggested that superoxide dismutase in tolerant genotypes provided first line protection from salt induced O2•-. Higher catalase (CAT) and ascorbate peroxidase (APX) played major role in H2O2 metabolism in tolerant genotypes. Both specific and in-gel activities of glutathione peroxidase (GPX) strongly proved the H2O2 metabolism for reducing oxidative damage in both tolerant and susceptible genotypes. However, higher peroxidase activity was important for mitigating salt stress in susceptible mungbean genotypes. Therefore, SOD, APX and GPX are very important for protecting salt mediated oxidative damage in mungbean genotype.
 Bangladesh J. Agril. Res. 44(3): 469-492, September 2019

Mung bean (Vigna radiata) cultivars mediated oviposition preference and development of Callosobruchus chinensis (Coleoptera: Chrysomelidae: Bruchinae)

The Azuki bean weevil, Callosobruchus chinensis (L.), is a destructive pest of stored mung bean [Vigna radiata (L.) Wilczek] as well as other leguminous seeds. The development of resistant seeds to manage this pest is of current great interest to plant breeders. In this study, we investigated the oviposition preference and development of C. chinensis on two susceptible mung bean cultivars (Seonhwa and Gyeongseon) and one previously reported resistant cultivar (Jangan), compared to the susceptible cowpea (Vigna unguiculata L.), cultivar (Yeonbun) using both multiple-choice and no-choice tests. In addition, the development of C. chinensis was also examined at four constant temperatures (20, 25, 30, and 35 °C). Both tests found cowpea to be the most suitable seed for oviposition. Total developmental time from oviposition to adult emergence ranged from 27.01 to 38.2 days, being shortest on cowpea and longest on the mung bean, cv. Jangan. However, no successful development of C. chinensis larvae on mung bean, cv. Jangan, occurred at any temperature. The highest rate of adult emergence and the longest adult longevity both occurred on cowpea and certain mung bean cultivars (Seonhwa and Gyeongseon), with the dramatic exception of cv. Jangan. These results suggest that the higher preference and performance of C. chinensis on cowpea (3.3 egg/seed) and least on mung bean, cv. Jangan (0.4 egg/seed). This information may facilitate the exploration of resistant genetic materials and chemicals associated with seeds for successful breeding. Further studies should examine the chemicals associated with mung bean cultivars and its resistant mechanism to develop a control method against bruchines.

Genomic and transcriptomic comparison of nucleotide variations for insights into bruchid resistance of mungbean (Vigna radiata [L.] R. Wilczek).

BackgroundMungbean (Vigna radiata [L.] R. Wilczek) is an important legume crop with high nutritional value in South and Southeast Asia. The crop plant is susceptible to a storage pest caused by bruchids (Callosobruchus spp.). Some wild and cultivated mungbean accessions show resistance to bruchids. Genomic and transcriptomic comparison of bruchid-resistant and -susceptible mungbean could reveal bruchid-resistant genes (Br) for this pest and give insights into the bruchid resistance of mungbean.ResultsFlow cytometry showed that the genome size varied by 61 Mb (mega base pairs) among the tested mungbean accessions. Next generation sequencing followed by de novo assembly of the genome of the bruchid-resistant recombinant inbred line 59 (RIL59) revealed more than 42,000 genes. Transcriptomic comparison of bruchid-resistant and -susceptible parental lines and their offspring identified 91 differentially expressed genes (DEGs) classified into 17 major and 74 minor bruchid-resistance–associated genes. We found 408 nucleotide variations (NVs) between bruchid-resistant and -susceptible lines in regions spanning 2 kb (kilo base pairs) of the promoters of 68 DEGs. Furthermore, 282 NVs were identified on exons of 148 sequence-changed-protein genes (SCPs). DEGs and SCPs comprised genes involved in resistant-related, transposable elements (TEs) and conserved metabolic pathways. A large number of these genes were mapped to a region on chromosome 5. Molecular markers designed for variants of putative bruchid-resistance–associated genes were highly diagnostic for the bruchid-resistant genotype.ConclusionsIn addition to identifying bruchid-resistance-associated genes, we found that conserved metabolism and TEs may be modifier factors for bruchid resistance of mungbean. The genome sequence of a bruchid-resistant inbred line, candidate genes and sequence variations in promoter regions and exons putatively conditioning resistance as well as markers detecting these variants could be used for development of bruchid-resistant mungbean varieties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0736-1) contains supplementary material, which is available to authorized users.

Multiple and mixed infections with yellow mosaic, leaf crinkle and bud necrosis disease complex in mungbean: A threat to cultivation of mungbean in India

The occurrence, incidence, yield losses and diagnostics of yellow mosaic disease (YMD) caused Mungbean yellow mosaic India virus (MYMIV), bud necrosis disease (BND) caused by Groundnut bud necrosis virus (GBNV) and Urdbean leaf crinkle disease complex (ULCD) were studied in mungbean (Vigna radiata) in Delhi condition. Eighteen mungbean cultivars were grown for successive four years from 2006 to 2009. Overall disease incidence of 11.6–18.5% for YMD; 14.4–20.5% for ULCD and 9.4–14.5% for BND were estimated. The mixed infection was studied in kharif season and overall it was 10.5% for YMD+ULCD, 8.3% for YMD+BND, 6.8% for ULCD+BND and 4.3% for YMD+ULCD+BND. None of the mungbean cultivars exhibited resistance to any of the three diseases. Yield loss estimated in susceptible mungbean cv. PS 16 was up to 61.1, 91.0 and 76.8% over control for YMD, ULCD and BND, respectively. Mixed infection with YMD and BND caused 92.8% yield losses. Biological and molecular detection using enzyme linked Immuno-sorbent assay (ELISA), polymerase chain reaction (PCR) and nucleotide analysis determined that MYMIV is the causal agent of YMD and GBNV of BND. Although etiology of ULCD was not confirmed, however, seed and sap transmission study showed that it is caused by an infectious agent.

Characterization of new sources of mungbean ( Vigna radiata (L.) Wilczek) resistance to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae)

The bruchid beetles Callosobruchus chinensis (L.) and C. maculatus (F.) are destructive pests of stored mungbean ( Vigna radiata (L.) Wilczek). The development of resistant mungbean varieties to manage bruchids is a major breeding objective. In the present study, we investigated the characteristics of resistance to C. chinensis and C. maculatus in two new resistant mungbean accessions V1128 and V2817, and two previously reported resistant accessions V2709 and V2802, compared to the susceptible mungbean cultivar, KPS1. Evaluation for resistance to both bruchid species using whole and decorticated seeds revealed that V1128 and V2817 were free from damage, V2709 and V2802 showed partial damage with low or moderate number of bruchids emerging from seeds, and KPS1 showed complete damage with the highest number of bruchids emerged. Comparison of the seeds harvested at seed filling, early maturity and full maturity stages revealed that the percentage of damaged seeds from resistant accessions was lower at all stages compared with KPS1. V1128 and V2817 showed complete resistance against both bruchids regardless of when their seeds were harvested, while resistance in V2709 and V2802 were most pronounced at full maturity, and KPS1 was totally damaged at all times tested. These results suggest that the chemical factor(s) conferring resistance is synthesized as early as the seed filling stage. Evaluation of resistance using artificial seeds showed that increasing the percentage of resistant seed powder adversely affected bruchid growth and development. The number of adults emerging from seeds and number of damaged seeds decreased while adult developmental period increased as the proportion of resistant seed powder increased. The weight of emerging male and female adults of C. maculatus was lighter than those from the seeds containing susceptible seed powder alone. However, C. chinensis adults were not affected by the same test. The results suggest that biochemical(s) in cotyledon tissue are responsible for the resistance and the seed coat had no protective role against the bruchids. Although all four resistant accessions evaluated are useful for mungbean breeding, V1128 and V2817 show complete resistance to both C. chinensis and C. maculatus. Thus, these two new resistant sources may be the most effective for breeding purposes.

Characterization of Callosobruchus chinensis (L.) resistance in Vigna umbellata (Thunb.) Ohwi & Ohashi

Resistance to azuki bean weevil, Callosobruchus chinensis, was studied in a series of field and laboratory experiments in two accessions of rice bean ( Vigna umbellata (Thunb.) Ohwi & Ohashi), one accession of black gram ( V. mungo (L.) Hepper), and one accession of mungbean, ( V. radiata (L.) Wilczek). Weevil damage to immature pods of the rice bean accessions, ‘Menaga’ and ‘Miyazaki’, was significantly less than to the susceptible mungbean, VC1973A. In mature pods, the pest damage to the pod wall of Menaga was significantly higher than to VC1973A, whereas the damage to Miyazaki was similar to VC1973A. Seeds within the pods of both rice bean accessions were resistant no matter when the pods were harvested. When the insects were exposed directly on dry seeds, both rice bean accessions and a black gram accession VM2164 were resistant to them. In artificial seeds made by mixing flour of the individual resistant Vigna accessions with VC1973A and subsequently exposed to bruchid oviposition, the higher the quantity of resistant Vigna flour the lower the number of bruchids that emerged from such seeds. No bruchids emerged from artificial seeds containing crude starch fraction from the three resistant Vigna accessions when such seeds were exposed to bruchid infestation, whereas many insects emerged from the seeds containing starch of VC1973A or flour of VC1973A alone. In artificial seeds made by mixing crude protein fractions of the three resistant Vigna accessions with flour of VC1973A, as the concentration of protein increased the number of C. chinensis adults that emerged decreased. Fractionation of crude proteins into acetone-precipitable proteins and peptide and amino acid portions resulted in the loss of antibiosis effect. Artificial seeds made from purified starch-polysaccharides fraction, however, exhibited antibiosis effects if prepared from the rice bean seed of Menaga and Miyazaki but not if made from the black gram seed, VM2164.

Proteome analysis of the relationship between bruchid-resistant and -susceptible mungbean genotypes

AbstractMungbean seeds are often attacked by bruchid insects causing serious damage to the grain during storage. The susceptible/resistant characteristics of mungbean against bruchids have been investigated at the protein level. Seeds of the susceptible cultivar Osaka-ryokuto and the resistant near-isogenic line BC20 were collected at 5, 9, 13, 17, 21 and 23 days after flowering. Crude, cytosol and membrane protein fractions were extracted from the seeds for twodimensional polyacrylamide gel electrophoresis analysis. In Osaka-ryokuto six proteins were found in higher amounts compared to BC20 in the crude protein extract. Some of these proteins were homologous to legumin B and liguleless1. And in BC20 one unique protein, homologous to SalF1R, was identified. In the subcellular protein fractions, nine unique proteins were identified in BC20, seven in the cytosol and two in the membrane fractions. These proteins were homologous to chitinase, alanine aminotransferase in membrane, and SalF1R, β-1,3- glucanase, protein phosphatase 2A, peroxidase BP 2A, provicilin and canavalin in the cytosol fractions. Previous findings of many researchers reveal that these are mainly defence-related proteins. Thus, it is suggested that the above proteins may play a role in maintaining the resistance of line BC20.

Morphological Characters in Vigna glabrescens Resistant to Agromyzids (Diptera: Agromyzidae)

Morphological characters associated with the infestation of bean fly, Ophiomyia Phaseoli (Tryon), and two other agromyzids, O. centrosematis (de Meijere) and Melanagromyza sojae (Zehntner), in one resistant Vigna glabrescens (Marechal, Mascherpa &; Strainier) accession, V 1160 (PI 207655), and one susceptible mungbean, Vigna radiate (L.) Wilczek, breeding line, VC 1973A, were investigated. The unifoliate leaves of V 1160 were significantly smaller and thinner than those of VC 1973A. The first trifoliate leaves of the former were significantly smaller and less pubescent than those of the latter. Accession V 1160 had significantly smaller and thinner petioles. The stems of V 1160 were glabrous and had shorter and thinner internodes than the stems of VC 1973A. Agromyzid adults preferred to visit VC 1973A plants and made more feeding/oviposition punctures in the leaves of this breeding line than in the same plant parts of V 1160. Consequently, significantly more bean fly larvae and pupae were found inside the stems of VC 1973A than in those of V 1160. This accession represents an important source for breeding mungbean cultivars resistant to agromyzids.

RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata, L. Wilczek)

Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F2 progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F2 population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.