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.


Introduction
Chickpea (Cicer arietinum L.) is an important pulse crop grown and consumed all over the world. As the world's population rises, the demand for grain legumes is also rising, and it is a permanent challenge to meet increasing demands. However, abiotic and biotic factors affect plant growth and pose a threat to sustainable agriculture and food production. Pathogens include fungi, bacteria, viruses, nematodes, and mycoplasma [1,2]. Several insect-transmitted viruses have been known to cause diseases in chickpea under field conditions: aphid-transmitted (virus in the families Bromoviridae, Luteoviridae, Nanoviridae, and Potyviridae) and leafhopper-transmitted (virus in the family Geminiviridae) viruses can lead to significant economic loss [3]. Among the leafhopper-transmitted viruses reported in chickpea, the most important and threatening viral disease is chickpea stunt disease (CSD).
CSD was recognized as a serious endemic problem in India as early as the 1970s [4]. The viruses, pea leaf roll virus in Iran [5]; subterranean clover red leaf virus (SCRLV), a strain of soybean dwarf virus, and beet western yellows virus (BWYV) in California [6,7]; as well as BWYV and bean leaf roll virus (BLRV) in Spain [8] were found to be associated with the chickpea stunt disease and discoloration symptoms. In India, BLRV was thought to be associated with the disease until 1993. CSD was first identified in India, and later the virus causing disease was identified as CpCDV and was shown to be transmitted in a persistent manner by the leafhopper O. albicinctus [9]. A survey showed CSD to be prevalent in the Indian states of Andhra Pradesh, Gujarat, Haryana, Madhya Pradesh, and Punjab, causing 75-95% losses in yield [10][11][12]. Later, Nahid et al. (2008) [13] in Pakistan and Kanakala et al.

Disease Symptoms
The symptoms of the disease caused by the dicot-infecting mastreviruses are yellowing, stunting, and dwarf symptoms in tobacco when infected by Tobacco yellow dwarf virus (TYDV) [19]. Bean yellow dwarf virus (BeYDV, now CpCDV-B [20]) in French bean causes stunting, chlorosis, and leaf curling symptoms [21]. The characteristic CSD symptoms are extreme stunting, shortening of internodes, reduction of leaf lamina, bushy and brittle appearance of plants, phloem browning in the collar region, leaf reddening in the case of indigenous types (desi), and yellowing in introduced (kabuli) types [9]. Field chickpea plants were found with symptoms like chlorosis, leaf smalling, and reddening of the chickpea leaves in Pakistan [13] and India [14] (Figure 1b,c). The yield loss is nearly total if the infection occurs in the early stage of growth; if infection occurs at the flowering stage, the yield loss is 75-90% [10]. viruses causing chickpea stunt in Africa and Asia, on the basis of 78% nucleotide identity in the genomic DNA and grouped all the South Asian mastreviruses as "Chickpea chlorotic dwarf virus". The species demarcation criteria of mastreviruses (www.ictvonline.org) are based on their nucleotide sequence identity, trans-replication of genomic components, capsid protein characteristics, transmitting vector species, natural host range, and symptom phenotype. To date, 19 strains of CpCDV (CpCDV A to S strains) have been reported in this genus. Until 1994, presence of CpCDV was limited to the Indian subcontinent, but they were later found in the Middle East and Africa. The emergence of new dicot-infecting mastreviruses have been variously reported in recent years (2013-2017) throughout South and North Africa, South Asia, and the Arabian Peninsula ( Figure 2) [13,14,[23][24][25][26][34][35][36][37].

Phylogenetic Relationships and Detection of Recombination
Dicot-infecting mastreviruses are widely distributed in the chickpea-growing regions of the world, including Australia, Africa, the Middle East, and South Asia. Genetic diversity based on the whole genome visualized two major groups, one with monocot-infecting mastreviruses and the other one comprises dicot-infecting mastreviruses (Figure 4). Among the dicot-infecting mastreviruses, two clades were visualized in phylogeny analysis, with one comprising dicot-infecting viruses from Africa, the Middle East, and South Asia (CpCDV-A to CpCDV-S), and the other clade consisting of dicot-infecting mastreviruses from Australia (CpRV, CpYV, TYDV-A, CpCAV, CpCV-A, CpCV-B, CpCV-C, CpCV-E, and CpCV-F) and Pakistan (CpYDV).  [11] reported that leafhopper O. orientalis (later names as O. albicinctus) successfully transmitted the CpCDV to a wide range of hosts belonging to the families Solanaceae, Leguminosae, and Chenopodiaceae, and they found that the virus was efficiently transmitted with a median acquisition access period (AAP), inoculation access period (IAP), and latency period (LP) of 8, 2.3, and 27.7 h, respectively. However, leafhopper transmission assays have not been conducted for all the hosts listed in Table 1 above. Therefore, more studies will reveal the efficacy of leafhoppers in transmitting CpCDV across multiple hosts.

Infectivity of Cloned Components
The family Geminiviridae consists of viruses which are transmitted by the vector, and most of them are not sap-transmitted, as the viruses are confined to phloem parenchymatous cells. In these cases, rubbing of the leaves with DNA does not work, as the viral DNA needs to reach the phloem tissue for its survival. This problem of virus delivery has been circumvented by Agrobacterium-mediated delivery of the viral genome.
N. benthamiana infected with clones CpCDV-A showed typical symptoms of yellowing, stunting, and crumpling of newly emerging leaves [24,55]. With CpCDV-B inoculation on N. benthamiana, N. tabacum, L. esculentum, D. stramonium, and A. thaliana plants became stunted, leaves developed interveinal chlorosis, and they exhibited severe downward curling symptoms [21]. Inoculation of CpCDV-C on N. benthamiana resulted in intense yellowing and downward leaf curling (Figure 3b) [14]. N. glutinosa showed severe stunting, small thick green leaves, and backward curling of apical leaves followed by a reduction in shoot elongation (Figure 3c) [14], and N. tabacum resulted in reduced apical leaves, dark green color, and downward leaf curling (Figure 3d) [14]. Young unfurling leaves became thick, dark green, and had mild backward leaf curling in CpCDV-C agroinoculated tomato plants (Figure 3e) [14]. Chickpea plants showed foliar yellowing and reduced leaf size, and plants were stunted [13].
In 2013, Kanakala et al. (2013) [56] observed differences in symptom phenotype when the viral genome was delivered through Agrobacterium in comparison with field infection. Kanakala et al. (2013) [56] showed the proliferation of axillary shoots with very small leaves, intense discoloration, and bushy stunted appearance of the plant as characteristic symptoms in both kabuli and desi genotypes tested. The reddening symptom seen in desi type in field conditions was not seen in agroinoculation. Interestingly, highly susceptible genotypes screened in this study dried after 25 days post inoculation (dpi). The death of virus infected chickpea plants was not observed under field conditions. The drying and death in agroinoculated plants might be due to the high concentration of viral inoculum introduced through direct Agrobacterium inoculations. CpCDV-C agroinoculated mustard (Family Brassicaceae) plants showed typical chlorosis, downward marginal folding, and were stunted (Figure 3f). Agroinoculated CpCDV-C in sesame (Family Pedaliaceae, variety Uma) produced very severe symptoms with thickening of leaves, downward folding, crumpling, and reduction of leaf lamina (Figure 3g). Similarly, CpCDV-A-agroinoculated watermelons showed yellowish/whitish areas or stripes in the flesh, which was discolored (i.e., orange instead of red) and, in some cases, displayed a clearly deformed shape [24].

Phylogenetic Relationships and Detection of Recombination
Dicot-infecting mastreviruses are widely distributed in the chickpea-growing regions of the world, including Australia, Africa, the Middle East, and South Asia. Genetic diversity based on the whole genome visualized two major groups, one with monocot-infecting mastreviruses and the other one comprises dicot-infecting mastreviruses (Figure 4). Among the dicot-infecting mastreviruses, two clades were visualized in phylogeny analysis, with one comprising dicot-infecting viruses from Africa, the Middle East, and South Asia (CpCDV-A to CpCDV-S), and the other clade consisting of dicot-infecting mastreviruses from Australia (CpRV, CpYV, TYDV-A, CpCAV, CpCV-A, CpCV-B, CpCV-C, CpCV-E, and CpCV-F) and Pakistan (CpYDV).
Mastreviruses are also well documented for having inter/intra species recombination [57,58]. Analyses of CpCDV sequences have suggested that recombination drives the evolution of this virus. The recombination analysis performed clearly indicates the presence of (a) inter-and intra-species recombination; (b) several breaking points within the Rep, CP, and intergenic common region (ICR); and (c) clear recombination breakpoints, hot and cold spots in Rep and CP genes, respectively [34,35,45]. These frequent exchanges of genomes might have resulted in the creation of new species and strains that may evolve to threaten agriculture. Mastreviruses are also well documented for having inter/intra species recombination [57,58]. Analyses of CpCDV sequences have suggested that recombination drives the evolution of this virus. The recombination analysis performed clearly indicates the presence of (a) inter-and intra-species recombination; (b) several breaking points within the Rep, CP, and intergenic common region (ICR); and (c) clear recombination breakpoints, hot and cold spots in Rep and CP genes, respectively  in X. strumarium. Similarly, CpCDV was also identified with CLCuBuV in cotton plants affected by leaf curl disease [47]. Similar to begomoviruses, some mastreviruses (i.e., Wheat dwarf India virus (WDIV) and CpCDV) have also been found to be associated with DNA satellite molecules in the field conditions [17,18]. More interestingly, βC1 has also been shown to be a pathogenicity determinant for both begomoviruses and monocot-infecting mastreviruses [59,60]. More recently, the association of CpCDV-C with Cotton leaf curl Multan betasatellite (CLCuMB) and Cotton leaf curl Multan alphasatellite (CLCuMA) was observed in spinach and its ability to trans-replicate CLCuMB in N. benthamiana was demonstrated [18].

Biology and Interaction of Begomoviruses and Satellite Molecules with CpCDV
However, our attempts to trans-replicate tomato leaf curl new Delhi virus (ToLCNDV) DNA B and betasatellite (CLCuMuB) by CpCDV-C in N. benthamiana showed only typical CpCDV symptoms and no trans-replication (Figure 3h,i). The presence of both ToLCNDV DNA B and CLCuMuB was not detected in molecular tests performed (data not shown). Similarly, co-inoculation of WDIV with CLCuMuB showed that CLCuMuB was not maintained by WDIV in wheat [17]. Since it is well known that geminivirus-encoded Rep protein binds to iterons, which plays a key role in initiating the replication of viral DNA [31], a specific betasatellite with compatible iteron-iteron-like sequences with CpCDV is needed to understand their interactions. It is still unknown whether CpCDV can trans-replicate CLCuMuB or any other betasatellite. Until now, there is no information available about the frequency of these associations in CpCDV epidemics.

Virus-Vector Interactions
Members of the genus Mastrevirus are transmitted by leafhoppers (family Cicadellidae). The leafhopper vector of the CpCDV causing the stunt disease in India was identified as O. albicinctus by Horn et al. (1994) [9]. Horn et al. (1993) [11] reported that leafhopper O. albicinctus successfully transmitted the CpCDV to a wide range of hosts belonging to the families Solanaceae, Leguminosae, and Chenopodiaceae, and they found that the virus was efficiently transmitted with a median acquisition access period (AAP), inoculation access period (IAP), and latency period (LP) of 8, 2.3, and 27.7 h, respectively. By serial transmission, they also showed that the vector can transmit the virus for most of their lifespan after a two-day AAP. There was similarity between CpCDV transmission with those conditions given for MSV [61] and BCTV [62][63][64]. More recently, Akhtar et al. (2011) [65] demonstrated that CpCDV is successfully transmitted by O. albicinctus. Its presence was detected in inoculated chickpea plants, and the vector was confirmed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test using specific polyclonal antibodies. Further studies on CpCDV-leafhopper transmission assays will reveal the alternative inoculum sources and CpCDV epidemics.

Detection and Diagnosis
The symptoms caused by mastreviruses in graminaceous hosts are often similar to symptoms caused by abiotic agents like nutritional deficiencies. In the case of dicot-infecting mastreviruses, it is difficult to distinguish a mastrevirus-infected plant from plants affected by other pathogens. In these circumstances, it is necessary to have virus-specific diagnostic reagents to detect the virus present in naturally infected plants. The diagnostics are also required to detect the virus in the insects visiting the plants in order to identity the vectors. Serological diagnostic methods such as DAS-ELISA, dot-blot ELISA, and tissue-blot immunoassay (TBIA) have been developed to detect the presence of CpCDV from infected field plant samples and viruliferous vector [10,46,65,66].
Over the past decade, rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP) has been extensively used to identify geminiviruses in most virus-infected plants. RCA was developed using the bacteriophage varphi 29 DNA polymerase, and was central in revolutionizing the detection and diagnosis of geminiviruses [67]. This technique is widely used to efficiently detect and characterize most of the dicot-infecting mastreviruses from field samples [13,14,34,35,45]. PCR-based methods involving gene-specific primers have also been developed and shown to efficiently detect CpCDV in infected plant tissues [13,14,46]. In addition, a pair of abutting primers have been utilized to amplify full-length dicot-infecting mastreviruses from Phi29 DNA polymerase-enriched DNA samples [45]. Kanakala et al. (2013) [56] detected CpCDV from field plants with a dot-blot hybridization method using a radiolabeled probe. Full-length replicative forms of CpCDV in agroinoculated plants were also detected using CpCDV-specific probes [13,14,18]. Recently, significant reductions in the costs of next-generation sequencing have accelerated use of deep sequencing for the detection and discovery of new strains of CpCDV [25,33]. Improved molecular techniques and whole-genome sequencing approaches for rapid detection of new viruses infecting chickpea offers new understanding of the evolution of CpCDV and its isolates.
All these molecular methods corroborate the importance of the extensive Mastrevirus diagnosis and control vector population to transmit disease. The relationships between mastreviruses infecting different host species need to be understood to develop management strategies that will prevent the further emergence of new viruses. An extensive global sampling and metagenomics analysis using next-generation sequencing of these viruses will identify the global diversity of dicot-infecting mastreviruses and inform better strategies for diagnostics and disease management.

Host Plant Resistance
In the past decade, an upsurge of chickpea viral diseases has been experienced, resulting in economic losses of chickpea production across the growing regions. Successful plant breeding programs for disease resistance depend on the successful identification of sources of resistance and the incorporation of resistance genes into commercial varieties [68,69]. Chickpea stunt disease is widespread in the old world, and causes considerable yield loss. The disease has been recognized as a serious challenge to chickpea cultivation, and resistance-breeding programs are being taken up. However, they are dependent on natural occurrences of the disease, as evaluation by inoculation through the vector is often cumbersome. At present, evaluation of CpCDV resistance is conducted on the basis of field screening of chickpea germplasm.
Among 10,000 germplasm lines screened for resistance to stunt disease, two lines (GG669 and ICCC10) were found to be field-resistant to CpCDV [12]. More recently, Kanakala et al. (2013) [56] has developed an agroinoculation technique to screen chickpea genotypes against CpCDV. This technique involves the construction of a complete tandem repeat CpCDV construct and the delivery of full-length CpCDV into germinated chickpea seed through Agrobacterium tumefaciens. Over 70 genotypes screened genotype SCGP-WR-29, which showed resistance in the field condition but exhibited 80% incidence under agroinoculation. Three agroinoculated genotypes (L-550, GNG-1499 (Gauri), and IPC 09-07) showed virus resistance and did not express any symptoms, and plants remained alive compared to susceptible genotypes. More interestingly, resistant plants were shown to be virus-free under PCR tests. These kinds of resistance screening tests have yet to be adopted to generate CpCDV-resistant cultivars on a wide scale. In the same study, an objective scoring to assess the response of chickpea genotypes to CSD by agroinoculation of CpCDV construct was also developed.

Genetic Engineering Approaches
RNA interference (RNAi) is a very promising strategy that has been employed to control both plant viruses and insect vectors [70,71]. Hairpin RNAi constructs containing sequences of CpCDV Rep and MP genes were stably expressed in N. benthamiana to provide immunity to CpCDV inoculation [72]. Baltes et al. (2015) [73] demonstrated a novel strategy for engineering resistance to BeYDV/CpCDV-B with a clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) prokaryotic immune system. Transgenic N. benthamiana plants expressing CRISPR-Cas reagents and challenged with BeYDV had reduced virus load and symptoms [73]. However, until now, there have been no reports on the transgenic control of any dicot-infecting Mastrevirus in chickpea. This will require significant progress in tissue culture and transformation technologies to CpCDV-resistant chickpea genotypes.
Similarly, RNAi has been successfully demonstrated in other leafhopper insect vectors. Silencing/knockdown of insect genes laccase-2/peptidoglycan recognition protein (PGRP-LC) resulted in significant mortality in leafhoppers [74,75]. Until now, there have been no RNAi-based silencing experiments studied in Mastrevirus insect vector. The identification of such candidate genes and the development of transgenic plants expressing dsRNA/SiRNA that target insect genes are necessary to control insect virus transmission under field conditions. Recent genome editing tools like CRISPR/Cas9 are highly suggested to modify virus/vector genes in order to develop effective resistance against CpCDV/O. albicinctus.

Future Prospects
CpCDV continues to be a threat to chickpea production worldwide. Although the virus was first reported in India in the year 1993, today CpCDV has been reported in Africa, the Middle East, and Australia, because of the polyphagous and widespread insect vector. At present, mixed infections, the emergence of new strains, and inter/intra recombinations among CpCDV strains/species might have increased its host range and caused new epidemics. Some major questions remain to be answered concerning (1) Mastrevirus-satellite interactions, (2) virus-host-insect interactions, (3) insect vector and its endosymbiont's efficacy in virus transmission, and (4) the discovery of CSD-resistant chickpea varieties. Considering the continuing new reports of CpCDV strains from new hosts and regions of the world, and given the importance of the fourth most widely grown pulse, continued research is needed to understand the biology, ecology, and epidemiology of CpCDV and its insect vector.
Over the past few years, a very low number of resistant chickpea varieties were screened through virus inoculations [12,56]. One possible new strategy that we can consider for engineering resistance against chickpea-infecting geminiviruses is genome editing through CRISPR/Cas9. Some recent studies exploited CRISPR/Cas9 technology, and could impart molecular immunity in single/mixed geminivirus infections by targeting the most conserved nonanucleotide sequence (TAATATTAC) present in the LIR or coding regions of the viral genome [73,[76][77][78]. Finally, pathogen-derived resistance strategies or gene editing methods need to be utilized to facilitate the development of chickpea cultivars with resistance to chickpea stunt disease.
Author Contributions: S.K. conceived the review structure and wrote the manuscript. Reviewing and Editing, S.K. and P.K.
Funding: This research received no external funding.