Serological and RT-PCR evaluation of African yam bean (Sphenostylis stenocarpa (Hochst ex. A. Rich) Harms) accessions to viral resistance under field condition.

African yam bean (AYB) (Sphenostylis stenocarpa (Hochst ex. A. Rich.) harms) an underutilized legume that produces nutritionally healthy seeds and tubers in some variety. The low yield of the crop is attributed to production constraints such as attacks by pest and disease-causing organisms such as fungi, bacteria and viruses. In this study, one hundred AYB accessions were evaluated for resistance to viral infection. The AYB accessions were planted using a randomized complete block design on the experimental field at the International Institute of Tropical Agriculture (IITA) Ibadan, Nigeria. Viral disease severity was assessed at 10, 12, 14, 16 and 18 weeks after planting (WAP) based on disease symptoms using disease severity index on visual scale of 1-5. Antigen-coated plate enzyme linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction were used to index diseased leaf samples collected from the field. Result from five virus species (Cowpea mild mottle virus, Cowpea mottle virus, Southern bean mosaic virus, Cowpea mosaic virus and Bean common mosaic virus) were detected in few accessions while mixed infections were observed in some accessions. TSs-552, TSs-577, TSs-580, TSs-560 and TSs-600 were devoid of viruses and could be resistant. There were no significant differences at p < 0.05 in the mean disease incidence (DI) of viral diseases. However, at 18 weeks after planting, TSs-604 had the highest (100%) mean DI while TSs-584 had the lowest (13.33%) mean DI. Cluster analysis based on the AUDPC produced 6 main clusters, the clusters revealed grouping patterns in which AYB lines with similar resistance ratings were shown to form unique clusters. The information generated from this study will contribute to the development of strategies in the management of virus diseases infecting AYB.

Molecular modelling of coat protein of the Groundnut bud necrosis tospovirus and its binding with Squalene as an antiviral agent: In vitro and in silico docking investigations.

Bud blight disease caused by groundnut bud necrosis virus (GBNV) is a serious constraint in the cultivation of agricultural crops such as legumes, tomato, chilies, potato, cotton etc. Owing to the significant damage caused by GBNV, an attempt was made to identify suitable organic antiviral agents through molecular modelling of the nucleocapsid Coat Protein of GBNV; molecular docking and molecular dynamics that disclosed the interaction of the ligands viz., Squalene and Ganoderic acid-A with coat protein of GBNV. Invitro inhibitory effect of Squalene and Ganoderic acid-A was examined in comparison with different concentrations, against GBNV in cowpea plants under glasshouse condition. The different concentrations of Squalene (50, 100, 150, 250 and 500 ppm) tested in vitro resulted in reduction of lesion numbers (1.69 cm2) as well as reduced virus titre in co-inoculation spray. The present study suggests the antiviral activity of Squalene by effectively fitting into binding site of coat protein of GBNV with favourable hydrophilic as well as strong hydrophobic interactions thereby challenging and blocking the binding of viral replication RNA with coat protein and propagation. The present organic antiviral molecules will be helpful in development of suitable eco-friendly formulations to mitigate GBNV infection disease in plants.

Genome characterization and diversity of trifolium virus 1: identification of a novel legume-infecting capulavirus.

A novel geminivirus was identified in France and Spain in asymptomatic plants of white clover (Trifolium repens) and shrub medick (Medicago arborea). Its genome has the hallmarks of a capulavirus, and its relationship to other capulaviruses was confirmed by phylogenetic analysis. White clover isolates formed a tight cluster in the phylogenetic tree, while shrub medick isolates formed two distinct, more divergent groups with sequence identity values close to the species cutoff. These three groups have likely participated in recombination events involving alfalfa leaf curl virus and French bean severe leaf curl virus. The name "trifolium virus 1" (TrV1) is proposed for this new Capulavirus. Three TrV1 genotypes (TrV1-A, TrV1-B, and TrV1-C) were clearly distinguished.

Two strains of a novel begomovirus encoding Rep proteins with identical ß1 strands but different ß5 strands are not compatible in replication.

Geminiviruses have genomes composed of single-stranded DNA molecules and encode a rolling-circle replication (RCR) initiation protein ("Rep"), which has multiple functions. Rep binds to specific repeated DNA motifs ("iterons"), which are major determinants of virus-specific replication. The particular amino acid (aa) residues that determine the preference of a geminivirus Rep for specific iterons (i.e., the trans-acting replication "specificity determinants", or SPDs) are largely unknown, but diverse lines of evidence indicate that most of them are closely associated with the so-called RCR motif I (FLTYP), located in the first 12-19 aa residues of the protein. In this work, we characterized two strains of a novel begomovirus, rhynchosia golden mosaic Sinaloa virus (RhGMSV), that were incompatible in replication in pseudorecombination experiments. Systematic comparisons of the Rep proteins of both RhGMSV strains in the DNA-binding domain allowed the aa residues at positions 71 and 74 to be identified as the residues most likely to be responsible for differences in replication specificity. Residue 71 is part of the ß-5 strand structural element, which was predicted in previous studies to contain Rep SPDs. Since the Rep proteins encoded by both RhGMSV strains are identical in their first 24 aa residues, where other studies have mapped potential SPDs, this is the first study lending direct support to the notion that geminivirus Rep proteins contain separate SPDs in their N-terminal domain.

Biology and genetic diversity of phasey bean mild yellows virus, a common virus in legumes in Australia.

This study examined the natural and experimental host range and aphid and graft transmission of the tentative polerovirus phasey bean mild yellows virus (PBMYV). Eleven complete coding sequences from PBMYV isolates were determined from a range of hosts and locations. We found two genetically distinct variants of PBMYV. PBMYV-1 was the originally described variant, and PBMYV-2 had a large putative recombination in open reading frame 5 such that PBMYV-1 and PBMYV-2 shared only 65-66% amino acid sequence identity in the P5 protein. The virus was transmitted by a clonal colony of cowpea aphids (Aphis craccivora) and by grafting with infected scions but was not transmitted by a clonal colony of green peach aphids (Myzus persicae). PBMYV was found in natural infections in 11 host species with a range of symptoms and severity, including seven important grain legume crops from across a wide geographic area in Australia. PBMYV was common and widespread in the tropical weed phasey bean (Macroptilium lathyroides), but it is likely that there are other major alternative hosts for the virus in temperate regions of Australia. The experimental host range of PBMYV included the Fabaceae hosts chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), and phasey bean, but transmissions failed to infect several other members of the families Asteraceae, Cucurbitaceae, Fabaceae and Solanaceae. PBMYV was commonly found in grain legume crops in eastern and western Australia, sometimes at greater than 90% incidence. This new knowledge about PBMYV warrants further assessments of its economic impact on important grain legume crops.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Viral infection can reduce the net nitrogen inputs of legume break crops and cover crops.

Legumes are used in crop rotations by both large-scale and smallholder farmers alike to increase soil fertility, especially before high-nitrogen-demanding crops such as corn (maize). Legume crop residues and green manures are rich in nitrogen due to mutualistic rhizobia, bacteria that live in their roots and convert atmospheric nitrogen into a biologically available form. Growers can obtain recommendations from local extension offices about how much less inorganic nitrogen fertilizer needs to be added to a subsequent crop following different legume break crops for the predominant soil type (the nitrogen fertilizer replacement value, or NFRV). Due to the intimate relationship between legumes and rhizobia, conditions that affect plant health can also affect the rhizobia and how much nitrogen they provide. We use a combination of empirical data and previously published values to estimate reductions in nitrogen inputs under outbreaks of plant viruses of varying severity. We also use historical fertilizer prices to examine the economic impacts of this lost fertilizer for farmers. We find that fertilizer losses are greatest for crops that fix large amounts of nitrogen, such as clover and alfalfa as opposed to common bean. The economic impact on farmers is controlled by the proportion of plants with viral infections and the price of synthetic fertilizer. In a year of high disease prevalence, attention is normally focused on the yield of the diseased crops. We suggest that farmers growing legumes as break crops should be concerned about yields of subsequent crops as well. Viral diseases can be difficult to diagnose in the field, so the easiest way for farmers to prevent unexpected yield losses in subsequent crops is to test their soil when it is feasible to do so.

Aphid transmission of nanoviruses.

The genus Nanovirus consists of plant viruses that predominantly infect legumes leading to devastating crop losses. Nanoviruses are transmitted by various aphid species. The transmission occurs in a circulative nonpropagative manner. It was long suspected that a virus-encoded helper factor would be needed for successful transmission by aphids. Recently, a helper factor was identified as the nanovirus-encoded nuclear shuttle protein (NSP). The mode of action of NSP is currently unknown in contrast to helper factors from other plant viruses that, for example, facilitate binding of virus particles to receptors within the aphids' stylets. In this review, we are summarizing the current knowledge about nanovirus-aphid vector interactions.

[Biological characteristics of bacteriophages infecting three typic rhizobia of legume]. / 侵染3æ ªæ¨¡å¼è±†ç§‘æ ¹ç˜¤èŒçš„å™¬èŒä½“ç”Ÿç‰©å­¦ç‰¹æ€§.

Bacteriophages infecting rhizobia of legume leads to a significant decrease in the number of rhizobia in soil and nodulation in legume, which finally affects nitrogen fixation and remarkably reduces crop yield. However, limited studies have focused on bacteriophages of rhizobia. In this study, three typic rhizobium strains of Bradyrhizobium diazoefficiens USDA110T, Sinorhizobium sojae CCBAU05684T and Sinorhizobium meliloti USDA1002T were used as the host bacteria. From each host, three rhizobiophages were isolated from an agricultural black soil with double-layer plate method. We isolated nine rhizobiophages and investigated their morphological feature and biological characteristics. The results showed that the phages of SMM (infecting Sinorhizobium meliloti USDA1002T) and BDM (infecting Bradyrhizobium diazoefficiens USDA110T) belonged to Myoviridae family, while phages of SSS (infecting Sinorhizobium sojae CCBAU05684T) belonged to Siphoviridae family. The optimal multiplicity of infection for nine phages ranged from 0.001 to 1.0. The one-step growth curve showed that the latent and rising periods of BDM were remarkably longer than that of SMM and SSS, but the bust size was minimal. Nine phages had the strongest infecting activity at 30-40 ℃ and at neutral pH condition. Comparative analysis showed that the biological characteristics of phages infected with the same host were different, with the differentiation being smaller than that of phages infected with different hosts.

Molecular characterization and analysis of conserved potyviral motifs in bean common mosaic virus (BCMV) for RNAi-mediated protection.

Australian bean common mosaic virus (BCMV) isolates were sequenced, and the sequences were compared to global BCMV and bean common mosaic necrosis virus (BCMNV) sequences and analysed for conserved potyviral motifs to generate in planta RNA-interference (RNAi) resistance. Thirty-nine out of 40 previously reported potyvirus motifs were conserved among all 77 BCMV/BCMNV sequences. Two RNAi target regions were selected for dsRNA construct design, covering 920 bp of the nuclease inclusion b (NIb) protein and 461 bp of the coat protein (CP). In silico prediction of the effectiveness of these constructs for broad-spectrum defence against the 77 BCMV and BCMNV sequences was done via analysis of putative 21-nucleotide (nt) and 22-nt small-interfering RNAs (siRNAs) generated from the target regions. The effectiveness of both constructs for siRNA generation and BCMV RNAi-mediated resistance was validated in Nicotiana benthamiana transient assays.

Cucumber mosaic virus Isolates from Greek Legumes are Associated with Satellite RNAs that are Necrogenic for Tomato.

Worldwide, Cucumber mosaic virus (CMV) is the causal agent of many economically important diseases. Based on immunological or molecular analysis, three distinct subgroups of CMV isolates can be identified (IA, IB, and II). In addition, some CMV isolates are associated with satellite RNAs (satRNAs), a type of noncoding transcript that may alter the symptoms of CMV infections. This study presents an analysis of CMV isolates occurring in legumes in Greece in respect to their genetic diversity, and the presence and diversity of their satRNA. Phylogenetic analysis of the CMV coat protein sequence of 18 legume and 5 tomato CMV isolates collected throughout Greece classified them within subgroups IA and IB, with a limited genetic diversity. The CMV satRNAs found in nine field legumes exhibiting mild symptoms and in one tomato with a necrotic syndrome contained a functional necrogenic motif; therefore, they were grouped within the necrogenic group of CMV-satRNAs. The necrotic phenotype was expressed in all legume CMV isolates containing necrogenic satRNAs when mechanically inoculated onto tomato plants. To our knowledge, this is the first observation that legumes host necrogenic CMV-satRNAs. The possible role of legumes in the epidemiology of CMV and necrogenic satRNA complex is discussed.

A novel endogenous badnavirus exists in Alhagi sparsifolia.

We report the recovery of a 7068-nt viral sequence from the "viral fossils" embedded in the genome of Alhagi sparsifolia, a typical desert plant. Although the full viral genome remains to be completed, the putative genome structure, the deduced amino acids and phylogenetic analysis unambiguously demonstrate that this viral sequence represents a novel species of the genus Badnavirus. The putative virus is tentatively termed Alhagi bacilliform virus (ABV). Southern blotting and inverse polymerase chain reaction (PCR) data indicate that the ABV-related sequence is integrated into the A. sparsifolia genome, and probably does not give rise to functional episomal virus. Molecular evidence that the ABV sequence exists widely in A. sparsifolia is also presented. To our knowledge, this is the first endogenous badnavirus identified from plants in the Gobi desert, and may provide new clues on the evolution, geographical distribution as well as the host range of the badnaviruses.

Development of rapid and highly sensitive detection of Bean common mosaic necrosis virus in leguminous crops using loop-mediated isothermal amplification assay.

Bean common mosaic necrosis virus (BCMNV) is a plant pathogenic virus that can infect leguminous crops such as kidney beans, sunn hemp, red beans, and mung beans. BCMNV has not been reported in Korea and is classified as a quarantine plant virus. Currently, the standard diagnostic method for diagnosis of BCMNV is reverse transcription (RT)-nested PCR system. However a more rapid monitoring system is needed to enable the testing of more samples. The use of highly efficient loop-mediated isothermal amplification (LAMP) assay for its detection has not yet been reported, and development of LAMP for detecting BCMNV in this study. In addition, confirmation of LAMP amplification can be achieved using restriction enzyme Mse I (T/TAA). The developed technique could be used for more rapid, specific and sensitive monitoring of BCMNV in leguminous crops than conventional nested RT-PCR.

Complete genome sequence of a new bipartite begomovirus infecting Macroptilium lathyroides in Brazil.

A distinct bipartite begomovirus was isolated in northeastern Brazil infecting Macroptilium lathyroides showing symptoms of yellow mosaic. The complete genome (DNA-A and DNA-B) of the virus was cloned using rolling circle amplification and subsequently sequenced. Clones presented the typical genomic organization of a New World bipartite begomovirus. Based on the current taxonomic criteria established for the genus Begomovirus, the virus corresponds to a new species, showing highest nucleotide identity with other Brazilian begomoviruses that infect leguminous hosts. In phylogenetic analysis the virus clustered with bean golden mosaic virus. Recombination events were not detected. We propose the name Macroptilium common mosaic virus (MacCMV) for the virus reported in this study.

Resistance to Bean common mosaic necrosis virus Conferred by the bc-1 Gene Affects Systemic Spread of the Virus in Common Bean.

Bean common mosaic necrosis virus (BCMNV) isolates belong to two pathogroups (PG), PG-III and PG-VI, which are distinguished in common bean due to the inability of the PG-III isolates of BCMNV to overcome the two recessive resistance alleles bc-1 and bc-12. The biological and molecular basis of this distinction between PG-III and PG-VI isolates of BCMNV is not known. Here, three isolates of BCMNV were typed biologically on a set of 12 bean differentials and molecularly through whole-genome sequencing. Two isolates (1755b and TN1a) were assigned to PG-VI and one isolate (NL8-CA) was assigned to PG-III. Isolate NL8-CA (PG-III) induced only local necrosis on inoculated leaves in 'Top Crop' and 'Jubila' bean harboring the I gene and the bc-1 allele, whereas isolates TN1, TN1a, and 1755b (all PG-VI) induced rapid whole-plant necrosis (WPN) in Top Crop 7 to 14 days postinoculation, and severe systemic necrosis but not WPN in Jubila 3 to 5 weeks postinoculation. In 'Redland Greenleaf C' expressing bc-1 and 'Redland Greenleaf B' expressing bc-12 alleles, isolate NL8-CA was able to systemically infect only a small proportion of upper uninoculated leaves (less than 13 and 3%, respectively). The whole genomes of isolates 1755b, TN1a, and NL8-CA were sequenced and sequence analysis revealed that, despite the overall high nucleotide sequence identity between PG-III and PG-VI isolates (approximately 96%), two areas of the BCMNV genome in the P1/HC-Pro and HC-Pro/P3 cistrons appeared to be more divergent between these two pathotypes of BCMNV. The data suggest that the phenotypic differences among PG-III and PG-VI isolates of BCMNV in common bean cultivars from host resistance groups 2, 3, and 9 carrying bc-1 alleles were related to the impaired systemic movement of the PG-III isolates to the upper, uninoculated leaves, and also suggest a role of the recessive bc-1 gene in interfering with systemic spread of BCMNV.

Functionally interchangeable cis-acting RNA elements in both genome segments of a picorna-like plant virus.

Cis-acting RNA structures in the genomes of RNA viruses play critical roles in viral infection, yet their importance in the bipartite genomes of the picorna-like, plant-infecting comoviruses has not been carefully investigated. We previously characterized SLC, a stem-loop structure in the 5' untranslated region (UTR) of the bean pod mottle comovirus (BPMV) RNA2, and found it to be essential for RNA2 accumulation in infected cells. Here we report the identification of SL1, a similar cis-acting element in the other BPMV genome segment - RNA1. SL1 encompasses a portion of RNA1 5' UTR but extends into the coding sequence for nine nucleotides, thus was missed in the previous study. While the stems of SL1 and SLC share little sequence similarity, their end loops are of the same size and identical for 11 of 15 nucleotides. Importantly, SL1 and SLC are functionally interchangeable, and separate exchanges of the stem and loop portions were likewise well tolerated. By contrast, the conserved loop sequence tolerated minimal perturbations. Finally, stem-loop structures with similar configurations were identified in two other comoviruses. Therefore, SL1 and SLC are likely essential comoviral RNA structures that play a conserved function in viral infection cycles.

Desmodium mottle virus, the first legumovirus (genus Begomovirus) from East Africa.

A novel bipartite legumovirus (genus Begomovirus, family Geminiviridae), that naturally infects the wild leguminous plant Desmodium sp. in Uganda, was molecularly characterized and named Desmodium mottle virus. The highest nucleotide identities for DNA-A, obtained from two field-collected samples, were 79.9% and 80.1% with the legumovirus, soybean mild mottle virus. DNA-B had the highest nucleotide identities (65.4% and 66.4%) with a typical non-legumovirus Old World begomovirus, African cassava mosaic virus. This is the first report of a legumovirus in East Africa and extends the known diversity of begomoviruses found infecting wild plants in this continent.

Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly.

Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean.

Molecular and biological properties of an endornavirus infecting winged bean (Psophocarpus tetragonolobus).

A double-stranded RNA (dsRNA) of approximately 15 kbp was isolated from asymptomatic winged bean (Psophocarpus tetragonolobus) plants. The size of the dsRNA, together with results of RT-PCR testing, suggested that it was the replicative form of a plant endornavirus. Cloning, sequencing, and sequence analyses confirmed the endornavirus nature of the dsRNA. Conserved motifs typical for endornaviruses were identified and their amino acid sequences compared with those of selected endornaviruses. Phylogenetic analyses revealed a close relationship with Bell pepper endornavirus, Phaseolus vulgaris endornavirus 2, and Hot pepper endornavirus. The dsRNA was present in most P. tetragonolobus genotypes tested. The virus was provisionally named Winged bean endornavirus 1 (WBEV-1).