Isolation of single-chain variable fragment (scFv) antibodies for detection of Chickpea chlorotic dwarf virus (CpCDV) by phage display.

Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus), has a wide host range and geographic distribution in many parts of the world, and it is one of the most important legume-infecting viruses. Detection of CpCDV-infected plants in the field and evaluation of viral resistance of plant cultivars are possible by conducting serological assays. Here, development and characterization of a specific recombinant monoclonal antibody for CpCDV as a diagnostic tool are described. For this purpose, the coat protein of CpCDV was expressed in Escherichia coli strain Rosetta (DE3) and used to screen a Tomlinson phage display antibody library to select a specific single-chain variable fragment (scFv). In each round of biopanning, the affinity of the phage for CpCDV-CP was tested by enzyme-linked immunosorbent assay (ELISA). The results showed that the specificity of the eluted phages increased after each round of panning. Testing of individual clones by ELISA showed that five clones of the monoclonal phage were more strongly reactive against CpCDV than the other clones. All selected positive clones contained the same sequence. The phage-displayed scFv antibody, which was named CpCDV-scFvB9, did not bind to other tested plant pathogens and showed high sensitivity in the detection of CpCDV. A Western blot assay demonstrated that CpCDV-scFvB9 reacted with the recombinant coat protein of CpCDV. Finally, the interaction CpCDV-scFvB9 and CpCDV-CP was analyzed in a molecular docking experiment. This is the first report on production of an scFv antibody against CpCDV, which could be useful for immunological detection of the virus.

Molecular characterization of Chickpea chlorotic dwarf virus strain D in Chickpea (Cicer arietinum) from District Dera Ismail Khan Khyber Pakhtunkhwa, Pakistan.

Chickpea chlorotic dwarf virus (CpCDV), a member of genus Mastrevirus (family Geminiviridae) is an important viral pathogen of chickpea and other legume crops in Middle East, North Africa, India and Pakistan. Among sixteen known strains of CpCDV three are known to infect legume crops in Punjab province of Pakistan. In this study diversity of CpCDV was explored in Khyber Pakhtunkhwa (KP) province of Pakistan. In year 2016, during a survey in the chickpea growing areas of district Dera Ismail Khan, CpCDV infected plants were identified. Leaf samples were collected, and a diagnostic PCR confirmed mastreivrus infection in 4 out of 100 samples. From these samples full-length genome of CpCDV was amplified using specific back-to-back primers. Virus molecules were sequenced to their entirety and sequence analysis of a molecule KRF4 (GenBank accession # KY952837) showed the highest pair wise sequence identity of 97% with a CpCDV molecule (KM229787) isolated from chickpea plant. An SDT analysis revealed it to be the D strain of CpCDV and a recombination detection program (RDP) showed it to be a recombinant between C (KM229780) and L (KT634301) strains of CpCDV. Thus, further supporting the intra-species recombination for CpCDV and presence of the same strain in chickpea growing areas of Pakistan other than Punjab province. This is the first identification of CpCDV (genus Mastrevirus family Geminiviridae) from chickpea (Cicer arietinum) plants in District Dera Ismail Khan, KP province, Pakistan.

Faba bean polerovirus 1 (FBPV-1); a new polerovirus infecting legume crops in Australia.

A new polerovirus species with the proposed name faba bean polerovirus 1 (FBPV-1) was found in winter legume crops and weeds in New South Wales, Australia. We describe the complete genome sequence of 5,631 nucleotides, containing all putative open reading frames, from two isolates, one from faba bean (Vicia faba) and one from chickpea (Cicer arietinum). FBPV-1 has a genome organization typical of poleroviruses with six open reading frames. However, recombination analysis strongly supports a recombination event in which the 5' portion of FBPV-1, which encodes for proteins P0, P1 and P1-P2, appears to be from a novel parent with a closest nucleotide identity of only 66% to chickpea chlorotic stunt virus. The 3' portion of FBPV-1 encodes for proteins P3, P4 and P3-P5 and shares 94% nucleotide identity to a turnip yellows virus isolate from Western Australia.

Chickpea chlorotic dwarf virus: An Emerging Monopartite Dicot Infecting Mastrevirus.

Chickpea stunt disease (CSD), caused by Chickpea chlorotic dwarf virus (CpCDV) is a threat to chickpea production leading to yield losses of 75⁻95%. Chickpea chlorotic dwarf virus is a monopartite, single-stranded circular DNA virus in the genus Mastrevirus and family Geminiviridae. It is transmitted by Orosius albicinctus in a circulative (persistent) and nonpropagative manner. Symptoms of CSD include very small leaves, intense discoloration (yellowing (kabuli type) and reddening (desi type)), and bushy stunted appearance of the plant. Presently, CpCDVs occurs in Africa, Asia, Australia, and the Middle East, causing extensive losses on economically important crops in in the families Fabaceae, Asteraceae, Amaranthaceae, Brassicaceae, Cucurbitaceae, Caricaceae, Chenopodiaceae, Leguminosae, Malvaceae, Pedaliaceae, and Solanaceae. High frequency of recombinations has played a significant role in the wide host range, diversification, and rapid evolution of CpCDVs. This review highlights the extensive research on the CpCDV genome diversity, host range, plant⁻virus⁻insect interactions, and RNA interference-based resistance of CpCDV, providing new insights into the host adaptation and virus evolution.

Shannon Entropy to Evaluate Substitution Rate Variation Among Viral Nucleotide Positions in Datasets of Viral siRNAs.

Next-generation sequencing has opened the door to the reconstruction of viral populations and examination of the composition of mutant spectra in infected cells, tissues, and host organisms. In this chapter we present details on the use of the Shannon entropy method to estimate the site-specific nucleotide relative variability of turnip crinkle virus, a positive (+) stranded RNA plant virus, in a large dataset of short RNAs of Cicer arietinum L., a natural reservoir of the virus. We propose this method as a viral metagenomics tool to provide a more detailed description of the viral quasispecies in infected plant tissue. Viral replicative fitness relates to an optimal composition of variants that provide the molecular basis of virus behavior in the complex environment of natural infections. A complete description of viral quasispecies may have implications in determining fitness landscapes for host-virus coexistence and help to design specific diagnostic protocols and antiviral strategies.

Molecular characterization of faba bean necrotic yellows viruses in Tunisia.

Faba bean necrotic yellows virus (FBNYV) (genus Nanovirus; family Nanoviridae) has a genome comprising eight individually encapsidated circular single-stranded DNA components. It has frequently been found infecting faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) in association with satellite molecules (alphasatellites). Genome sequences of FBNYV from Azerbaijan, Egypt, Iran, Morocco, Spain and Syria have been determined previously and we now report the first five genome sequences of FBNYV and associated alphasatellites from faba bean sampled in Tunisia. In addition, we have determined the genome sequences of two additional FBNYV isolates from chickpea plants sampled in Syria and Iran. All individual FBNYV genome component sequences that were determined here share > 84% nucleotide sequence identity with FBNYV sequences available in public databases, with the DNA-M component displaying the highest degree of diversity. As with other studied nanoviruses, recombination and genome component reassortment occurs frequently both between FBNYV genomes and between genomes of nanoviruses belonging to other species.

New strains of chickpea chlorotic dwarf virus discovered on diseased papaya and tomato plants in Burkina Faso.

This is the first description of full genome sequences of chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus; family Geminiviridae) identified in papaya and tomato plants sampled in Burkina Faso. The CpCDV full genome sequences from papaya and tomato share the highest pairwise sequence identity (84% and 93.5%) with Sudanese isolates of the CpCDV-K and CpCDV-M strains, respectively. Based on the strain demarcation threshold (>94% identity) for mastreviruses, we propose two new strains, CpCDV-Q and CpCDV-R, identified in papaya and tomato, respectively. Phylogenetic analysis confirmed that the sequences belong to a distinct clade of the highly diverse population of CpCDVs. Evidence of inter-strain recombination provided more support for the important role of recombination in CpCDV evolution. The discovery of CpCDV on papaya, a previously unsuspected host, raises many questions about the natural and potential host range of this dicot-infecting mastrevirus species that is reported to be emerging worldwide.

Molecular diversity of Chickpea chlorotic dwarf virus in Sudan: high rates of intra-species recombination - a driving force in the emergence of new strains.

In Sudan Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae) is an important pathogen of pulses that are grown both for local consumption, and for export. Although a few studies have characterised CpCDV genomes from countries in the Middle East, Africa and the Indian subcontinent, little is known about CpCDV diversity in any of the major chickpea production areas in these regions. Here we analyse the diversity of 146 CpCDV isolates characterised from pulses collected across the chickpea growing regions of Sudan. Although we find that seven of the twelve known CpCDV strains are present within the country, strain CpCDV-H alone accounted for ∼73% of the infections analysed. Additionally we identified four new strains (CpCDV-M, -N, -O and -P) and show that recombination has played a significant role in the diversification of CpCDV, at least in this region. Accounting for observed recombination events, we use the large amounts of data generated here to compare patterns of natural selection within protein coding regions of CpCDV and other dicot-infecting mastrevirus species.

Identification of an Australian-like dicot-infecting mastrevirus in Pakistan.

Although members of five distinct viral species in the genus Mastrevirus (family Geminiviridae) infect dicotyledonous plants in Australia, in the remainder of the world, only a single dicot-infecting mastrevirus, chickpea chlorotic dwarf virus (CpCDV) has ever been identified. This virus has been found infecting leguminous hosts in Africa, the Middle East and the Indian subcontinent. To further explore the diversity of CpCDV in Pakistan, ten full mastrevirus genome sequences from chickpea and lentil plants were determined. Eight of these genomes were from previously described CpCDV strains and included the first reported strain D and H isolates in Pakistan. Two other genomes derived from infected chickpea plants are more closely related to dicot-infecting mastreviruses found in Australia than they are to CpCDV. These two divergent genomes shared less than 75 % genome-wide nucleotide sequence identity with other characterised mastreviruses and therefore are likely to belong to a second species of dicot-infecting mastreviruses outside of Australia. We propose naming this species Chickpea yellow dwarf virus. We discuss how the presence of chickpea yellow dwarf virus (CpYDV) in Pakistan weakens the hypothesis that Australia is the geographical origin of the dicot-infecting mastreviruses.

Response of chickpea genotypes to Agrobacterium-mediated delivery of Chickpea chlorotic dwarf virus (CpCDV) genome and identification of resistance source.

Chickpea stunt disease caused by Chickpea chlorotic dwarf virus (CpCDV) (genus Mastrevirus, family Geminiviridae) is the most important biotic stress affecting chickpea crops worldwide. A survey conducted on the incidence of stunt disease clearly revealed high incidence of the disease with severe symptom expression in both indigenous and imported genotypes. To manage the disease in a sustainable way, resistant genotypes need to be bred by adopting objective and precise assessment of the disease response of chickpea genotypes. At present, evaluation of CpCDV resistance is conducted on the basis of natural infection in the field, which is bound to be erroneous due to vagaries in vector population. To circumvent the above problems, we devised an agroinoculation technique that involves the delivery of viral genomic DNA through Agrobacterium tumefaciens. An objective scoring system assigning quantitative value to different symptoms has been evolved to assess the response of chickpea genotypes to CpCDV inoculation. Using the inoculation and scoring techniques, we screened 70 genotypes, which helped in differentiating field resistance that is more due to resistance to vector feeding than resistance to the virus.

Analysis of the sequence of a dicot-infecting mastrevirus (family Geminiviridae) originating from Syria.

Chickpea stunt disease (CSD) across southern Asia, the Middle East and North Africa is caused by a number of viruses that include single-stranded DNA viruses of the genus Mastrevirus (family Geminiviridae). Despite the importance of CSD in reducing chickpea and lentil production, until recently little was known of the nature of the pathogens causing the disease. Sequence characterisation of virus isolates from Sudan and Pakistan showed the viruses concerned to potentially be new mastrevirus species related to Bean yellow dwarf virus (BeYDV), a virus known to occur in both southern Africa and southern Asia. Here we have determined the complete nucleotide sequence of a mastrevirus associated with CSD in Syria. This virus represents a proposed new species, closely related to the recently characterised Chickpea chlorotic dwarf Sudan virus and Chickpea chlorotic dwarf Pakistan virus but with the highest sequence identity to BeYDV, for which we propose the name Chickpea chlorotic dwarf Syria virus. In addition the biological integrity of the clone was confirmed by infection of Nicotiana benthamiana plants using Agrobacterium-mediated inoculation.

Two novel mastreviruses from chickpea (Cicer arietinum) in Australia.

Two novel mastreviruses (genus Mastrevirus; family Geminiviridae), with proposed names chickpea chlorosis virus (CpCV) and chickpea redleaf virus, are described from chickpea (Cicer arietinum) from eastern Australia. The viruses have genomes of 2,582 and 2,605 nucleotides, respectively, and share similar features and organisation with typical dicot-infecting mastreviruses. Two distinct strains of CpCV were suggested by phylogenetic analysis. Additionally, a partial mastrevirus Rep sequence from turnip weed (Rapistrum rugosum) indicated the presence of a distinct strain of Tobacco yellow dwarf virus (TYDV). In phylogenetic analyses, isolates of Bean yellow dwarf virus, Chickpea chlorotic dwarf Pakistan virus and Chickpea chlorotic dwarf Sudan virus from southern and northern Africa and south-central and western Asia clustered separately from these three viruses from Australia. An Australian, eastern Asian, or south-eastern Asian origin for the novel mastreviruses and TYDV is discussed.

Inactivation of baculovirus by isoflavonoids on chickpea (Cicer arietinum) leaf surfaces reduces the efficacy of nucleopolyhedrovirus against Helicoverpa armigera.

Biological pesticides based on nucleopolyhedroviruses (NPVs) can provide an effective and environmentally benign alternative to synthetic chemicals. On some crops, however, the efficacy and persistence of NPVs is known to be reduced by plant specific factors. The present study investigated the efficacy of Helicoverpa armigera NPV (HearNPV) for control of H. armigera larvae, and showed that chickpea reduced the infectivity of virus occlusion bodies (OBs) exposed to the leaf surface of chickpea for at least 1 h. The degree of inactivation was greater on chickpea than that previously reported on cotton, and the mode of action is different from that of cotton. The effect was observed for larvae that consumed OBs on chickpea leaves, but it also occurred when OBs were removed after exposure to plants and inoculated onto artificial diet, indicating that inhibition was leaf surface-related and permanent. Despite their profuse exudation from trichomes on chickpea leaves and their low pH, organic acids-primarily oxalic and malic acid-caused no inhibition. When HearNPV was incubated with biochanin A and sissotrin, however, two minor constituents of chickpea leaf extracts, OB activity was reduced significantly. These two isoflavonoids increased in concentration by up to 3 times within 1 h of spraying the virus suspension onto the plants and also when spraying only the carrier, indicating induction was in response to spraying and not a specific response to the HearNPV. Although inactivation by the isoflavonoids did not account completely for the level of effect recorded on whole plants, this work constitutes evidence for a novel mechanism of NPV inactivation in legumes. Expanding the use of biological pesticides on legume crops will be dependent upon the development of suitable formulations for OBs to overcome plant secondary chemical effects.