A viral small interfering RNA-host plant mRNA pathway modulates virus-induced drought tolerance by enhancing autophagy.

Virus-induced drought tolerance presents a fascinating facet of biotic-abiotic interaction in plants, yet its molecular intricacies remain unclear. Our study shows that cowpea mild mottle virus (CPMMV) infection enhances drought tolerance in common bean (Phaseolus vulgaris) plants through a virus-derived small interfering RNA (vsiRNA)-activated autophagy pathway. Specifically, a 21 nt vsiRNA originating from the CPMMV Triple Gene Block1 (TGB1) gene targeted the 5' untranslated region (UTR) of the host Teosinte branched 1, Cycloidea, Proliferating Cell Factor (TCP) transcription factor gene PvTCP2, independent of the known role of TGB1 as an RNA silencing suppressor. This targeting attenuated the expression of PvTCP2, which encodes a transcriptional repressor, and in turn upregulated the core autophagy-related gene (ATG) PvATG8c, leading to activated autophagy activity surpassing the level induced by drought or CPMMV infection alone. The downstream EARLY RESPONSIVE TO DEHYDRATION (ERD) effector PvERD15 is a homologue of Arabidopsis thaliana AtERD15, which positively regulates stomatal aperture. PvERD15 was degraded in PvATG8c-mediated autophagy. Therefore, we establish a TGB1-PvTCP2-PvATG8c-PvERD15 module as a trans-kingdom fine-tuning mechanism that contributes to virus-induced drought tolerance in plant-drought-virus interactions.

Cowpea lipid transfer protein 1 regulates plant defense by inhibiting the cysteine protease of cowpea mosaic virus.

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

Arabidopsis latent virus 1, a comovirus widely spread in Arabidopsis thaliana collections.

Transcriptome studies of Illumina RNA-Seq datasets of different Arabidopsis thaliana natural accessions and T-DNA mutants revealed the presence of two virus-like RNA sequences which showed the typical two-segmented genome characteristics of a comovirus. This comovirus did not induce any visible symptoms in infected A. thaliana plants cultivated under standard laboratory conditions. Hence it was named Arabidopsis latent virus 1 (ArLV1). Virus infectivity in A. thaliana plants was confirmed by quantitative reverse transcription polymerase chain reaction, transmission electron microscopy and mechanical inoculation. Arabidopsis latent virus 1 can also mechanically infect Nicotiana benthamiana, causing distinct mosaic symptoms. A bioinformatics investigation of A. thaliana RNA-Seq repositories, including nearly 6500 Sequence Read Archives (SRAs) in the NCBI SRA database, revealed the presence of ArLV1 in 25% of all archived natural A. thaliana accessions and in 8.5% of all analyzed SRAs. Arabidopsis latent virus 1 could also be detected in A. thaliana plants collected from the wild. Arabidopsis latent virus 1 is highly seed-transmissible with up to 40% incidence on the progeny derived from infected A. thaliana plants. This has probably led to a worldwide distribution in the model plant A. thaliana with as yet unknown effects on plant performance in a substantial number of studies.

Isolation of Cowpea Mosaic Virus-Binding Peptides.

The plant virus cowpea mosaic virus (CPMV) is a natural nanocarrier that has been developed as a platform technology for the delivery of various payloads including peptide epitopes for vaccines, contrast agents for imaging, and drugs for therapy. Genetic fusion and chemical conjugations are the mainstay approaches to load the active ingredient to the exterior and/or interior of CPMV. However, these methods have limitations; genetic engineering is limited to biologics, and chemical alteration often requires multistep reactions with modification of both CPMV and the active ingredient. Either method can also result in particle instability. Therefore, to provide an alternate path toward CPMV functionalization, we report the isolation of peptides that specifically bind to CPMV, termed CPMV-binding peptides (CBP). We used a commercial M13 phage display 7-mer peptide library to pan for and select peptides that selectively bind to CPMV. Biopanning and characterization of lead candidates resulted in isolation of the motif "GWRVSEF/L" as the CPMV-specific motif with phenylalanine (F) at the seventh position being stronger than leucine (L). Specificity to CPMV was demonstrated, and cross-reactivity toward other plant viruses was not observed. To demonstrate cargo loading, GWRVSEF was tagged with biotin, fluorescein isothiocyanate (FITC), and a human epidermal growth factor receptor 2 (HER2)-specific targeting peptide ligand. Display of the active ingredient was confirmed, and utility of tagged and targeted CPMV in cell binding assays was demonstrated. The CBP functionalization strategy offers a new avenue for CPMV nanoparticle functionalization and should offer a versatile tool to add active ingredients that otherwise may be difficult to conjugate or display.

First complete genome sequence of an isolate of cowpea severe mosaic virus from South America.

In this study, the complete nucleotide sequence of a Brazilian isolate of cowpea severe mosaic virus (CPSMV) is presented for the first time. To date, the CPSMV-DG isolate, from the USA, is the only one with the complete known genome. High-throughput sequencing (Illumina HiSeq) and Sanger sequencing of the total RNA extract from a cowpea plant collected in Teresina city, Brazil, revealed the genome sequence of the CPSMV-Ter1 isolate. RNA-1 and RNA-2 are, respectively, 5921 and 3465 nucleotides (nt) long without the poly(A) tail, and show 77.91% and 76.08% nt sequence identity with CPSMV-DG, considered the type isolate of the species. The open reading frames (ORFs) were determined and the cleavage sites of the polyproteins were predicted. Although the two isolates show a similar genomic organization, there was a low percentage of sequence identity between Ter1 and DG. Furthermore, pairwise comparisons of a partial RNA-1 fragment between CPSMV-Ter1 and 11 CPSMV isolates from Brazil indicated 94.6 to 94.8% nt and 98.9% to 99.4% aa sequence identities.

Complete genome sequence of a novel comovirus infecting common bean.

The complete genome sequence of a novel comovirus identified in Guanajuato, Mexico, in a common bean plant (Phaseolus vulgaris L.) coinfected with Phaseolus vulgaris alphaendornavirus 1 (PvEV-1) and Phaseolus vulgaris alphaendornavirus 2 (PvEV-2) is presented. According to the current ICTV taxonomic criteria, this comovirus corresponds to a new species, and the name "Phaseolus vulgaris severe mosaic virus" (PvSMV) is proposed for this virus based on the observed symptoms of "severe mosaic" syndrome caused by comoviruses in common bean. PvSMV is closely related to bean pod mosaic virus (BPMV), and its genome consists of two polyadenylated RNAs. RNA-1 (GenBank accession number MN837498) is 5969 nucleotides (nt) long and encodes a single polyprotein of 1856 amino acids (aa), with an estimated molecular weight (MW) of 210 kDa, that contains putative proteins responsible for viral replication and proteolytic processing. RNA-2 (GenBank accession number MN837499) is 3762 nt long and encodes a single polyprotein of 1024 aa, with an estimated MW of 114 kDa, that contains putative movement and coat proteins. Cleavage sites were predicted based on similarities in size and homology to aa sequences of other comoviruses available in the GenBank database. Symptoms associated with PvSMV include mosaic, local necrotic lesions, and apical necrosis. This is the first report of a comovirus infecting common bean in Mexico.

Freeze-Drying To Produce Efficacious CPMV Virus-like Particles.

In situ cancer vaccination that uses immune stimulating agents is revolutionizing the way that cancer is treated. In this realm, viruses and noninfectious virus-like particles have gained significant traction in reprogramming the immune system to recognize and eliminate malignancies. Recently, cowpea mosaic virus-like particles (VLPs) have shown exceptional promise in their ability to fight a variety of cancers. However, the current methods used to produce CPMV VLPs rely on agroinfiltration in plants. These protocols remain complicated and labor intensive and have the potential to introduce unwanted immunostimulatory agents, like lipopolysaccharides. This Letter describes a simple "post-processing" method to remove RNA from wild-type CPMV, while retaining the structure and function of the capsid. Lyophilization was able to eject encapsulated RNA to form lyo-eCPMV and, when purified, eliminated nearly all traces of encapsulated RNA. Lyo-eCPMV was characterized by cryo-electron microscopy single particle reconstruction to confirm the structural integrity of the viral capsid. Finally, lyo-eCPMV showed  equivalent anticancer efficacy as eCPMV, produced by agroinfiltration, when using an invasive melanoma model. These results describe a straightforward method to prepare CPMV VLPs from infectious virions.

Amino acids at the exposed C-terminus of the S coat protein of cowpea mosaic virus play different roles in particle formation and viral systemic movement.

The icosahedral capsid of cowpea mosaic virus is formed by 60 copies of the large (L) and small (S) coat protein subunits. The 24-amino-acid C-terminal peptide of the S coat protein can undergo proteolytic cleavage without affecting particle stability or infectivity. Mutagenic studies have shown that this sequence is involved in particle assembly, virus movement, RNA encapsidation and suppression of gene silencing. However, it is unclear how these processes are related, and which part(s) of the sequence are involved in each process. Here, we have analysed the effect of mutations in the C-terminal region of the S protein on the assembly of empty virus-like particles and on the systemic movement of infectious virus. The results confirmed the importance of positively charged amino acids adjacent to the cleavage site for particle assembly and revealed that the C-terminal 11 amino acids are important for efficient systemic movement of the virus.

Genome sequence and phylogenetic analysis of a novel comovirus from tabasco pepper (Capsicum frutescens).

A virus isolate from tabasco pepper (Capsicum frutescens) has been reported as a strain of the comovirus Andean potato mottle virus (APMoV). Using the replicative intermediate viral dsRNA, the pepper virus strain was sequenced by Illumina MiSeq. The viral genome was de novo assembled resulting in two RNAs with lengths of 6028 and 3646 nt. Nucleotide sequence analysis indicated that they corresponded to the RNA-1 and RNA-2 of a novel comovirus which we tentatively named pepper mild mosaic virus (PepMMV). Predictions of the open reading frame (ORF) of RNA-1 resulted in a single ORF of 5871 nt with five cistrons typical of comoviruses, cofactor proteinase, helicase, viral protein genome-linked, 3C-like proteinase (Pro), and RNA-dependent RNA polymerase (RdRP). Similarly, sequence analysis of RNA-2 resulted in a single ORF of 3009 nt with two cistrons typical of comoviruses: movement protein and coat protein (large coat protein and small coat proteins). In pairwise amino acid sequence alignments using the Pro-Pol protein, PepMMV shared the closest identities with broad bean true mosaic virus and cowpea mosaic virus, 56% and 53.9% respectively. In contrast, in alignments of the amino acid sequence of the coat protein (small and large coat proteins) PepMMV shared the closest identities to APMoV and red clover mottle virus, 54% and 40.9% respectively. A phylogenetic tree constructed using the conserved domains for the Pro-Pol from all members of the family Secoviridae confirmed the comovirus nature of the virus. Phylogenetic and sequence analyses supports proposing PepMMV as a new species of the genus Comovirus.

A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules.

Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chemical synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small molecules for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid production is estimated to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technology for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodology we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entrée to suites of molecules, some new-to-nature, that are recalcitrant to chemical synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biology, this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines.

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.

Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum.

Pea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus-induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)-derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4-ß-D-glucanase, respectively. The features of the 'one-step' BPMV-derived VIGS vector include (i) the ease of rub-inoculation, without any need for biolistic or agro-inoculation procedures, (ii) simple cost-effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low- to high-throughput VIGS studies.

Proteomic analysis of the plasma membrane-movement tubule complex of cowpea mosaic virus.

Cowpea mosaic virus forms tubules constructed from the movement protein (MP) in plasmodesmata (PD) to achieve cell-to-cell movement of its virions. Similar tubules, delineated by the plasma membrane (PM), are formed protruding from the surface of infected protoplasts. These PM-tubule complexes were isolated from protoplasts by immunoprecipitation and analysed for their protein content by tandem mass spectrometry to identify host proteins with affinity for the movement tubule. Seven host proteins were abundantly present in the PM-tubule complex, including molecular chaperonins and an AAA protein. Members of both protein families have been implicated in establishment of systemic infection. The potential role of these proteins in tubule-guided cell-cell transport is discussed.

Complete genome sequence of bean rugose mosaic virus, genus Comovirus.

Since the first report in Costa Rica in 1971, bean rugose mosaic virus (BRMV) has been found in Colombia, El Salvador, Guatemala and Brazil. In this study, the complete genome sequence of a soybean isolate of BRMV from Paraná State, Brazil, was determined. The BRMV genome consists of two polyadenylated RNAs. RNA1 is 5909 nucleotides long and encodes a single polypeptide of 1856 amino acids (aa), with an estimated molecular weight of 210 kDa. The RNA1 polyprotein contains the polypeptides for viral replication and proteolytic processing. RNA2 is 3644 nucleotides long and codes for a single polypeptide of 1097 aa, containing the movement and coat proteins. This is the first complete genome sequence of BRMV. When compared with available aa sequences of comoviruses, the highest identities of BRMV coat proteins and proteinase polymerase were 57.5 and 58 %, respectively. These were below the 75 and 80 % identity limits, respectively, established for species demarcation in the genus. This confirms that BRMV is a member of a distinct species in the genus Comovirus.

Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification.

Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.

Rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of M2 protein linked to flagellin in plants using viral vectors.

BACKGROUND: The extracellular domain of matrix protein 2 (M2e) of influenza A virus is a promising target for the development of a universal vaccine against influenza because M2e sequences are highly conserved among human influenza A strains. However, native M2e is poorly immunogenic, but its immunogenicity can be increased by delivery in combination with adjuvants or carrier particles. It was previously shown that fusion of M2e to bacterial flagellin, the ligand for Toll-like receptor (TLR) 5 and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of M2e. RESULTS: In this study, we report for the first time the transient expression in plants of a recombinant protein Flg-4M comprising flagellin of Salmonella typhimurium fused to four tandem copies of the M2e peptide. The chimeric construct was expressed in Nicotiana benthamiana plants using either the self-replicating potato virus X (PVX) based vector, pA7248AMV-GFP, or the cowpea mosaic virus (CPMV)-derived expression vector, pEAQ-HT. The highest expression level up to 30% of total soluble protein (about 1 mg/g of fresh leaf tissue) was achieved with the PVX-based expression system. Intranasal immunization of mice with purified Flg-4M protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza virus. CONCLUSIONS: This study confirms the usefulness of flagellin as a carrier of M2e and its relevance for the production of M2e-based candidate influenza vaccines in plants.

The bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation.

UNLABELLED: Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction. IMPORTANCE: Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus.

Fine-tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system.

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA-2, termed CPMV-HT, in which the sequence to be expressed is positioned between a modified 5' UTR and the 3' UTR has been successfully used for the plant-based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5' UTR can dramatically influence expression levels, the role of the 3' UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3'UTR of CPMV RNA-2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress-HT-GFP. The results showed that the presence of a 3' UTR in the CPMV-HT system is important for achieving maximal expression levels. Removal of the entire 3' UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y-shaped secondary structure formed by nucleotides 125-165 of the 3' UTR plays a key role in its function; mutations that disrupt this Y-shaped structure have an effect equivalent to the deletion of the entire 3' UTR. Our results suggest that the Y-shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5' and 3' UTRs in CPMV-HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.

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.

Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design.

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105-106 times more sensitive than ELISA and 100-1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.