IntroductionAbelmoschusesculentus (family: Malvaceae) otherwise known as bhendi, okra or lady’s finger is previously included in the genus Hibiscus; however,now it is recognized as a distinct genus ‘Abelmoschus’ due to itscaducous nature of the calyx (Dhankhar et al., 2005). The word ‘Abelmoschus’is originated from the Arabian word “abul-l-mosk” meaning “source ofmusk,” denoting to the musky smell of theseeds. Bhendi is widely cultivatedin the temperature regions of Asia, Africa andSouthern Europe (Charrier, 1984). Thisplant is thought to be the native of South Africa and the first recordedreference was from Egyptians in 1216 A.D (Lamont, 1999). However, ten species of bhendi plants are present in India, of which A.
esculentusis the only cultivated species (Dhankar et al. 2005). Bhendi is an allopolyploid with the lowest chromosomenumber in A. angulus (2n=56) and thehighest in A.
caillei (2n=100), whichis an amphiployploid between A. esculentus (2n=13-140) and A. manihot(2n=60-68) (Siemonsma, 1982). Developing countries like Asia and Africacontribute more than 99% of okra cultivation compared with other parts of theworld. Globally, bhendi cultivation is occupying an area of 1.
83 million withthe annual yield of 9.62 million metric tons (MT). India contributes first inthe world with 6.5 million metric tons (72% of the total world production) of bhendiproduced from over 0.5 million hectare land (FAOSTAT, 2014). Bhendi is a multipurpose crop withhuge economical importance. The green immature pod is a rich source of vitamins,iron, dietary fibers, amino acids (lysine and tryptophan), potassium, calcium,magnesium, and other mineral constituents (Hughes, 2009). Its seeds are a richsource of oil (30-40%) and proteins (15-20%) (Gemede et al.
2015). Its matureseed and stems are ample source of crude fibre and used in paper industry (Kochlar, 1986). The roots and stems are used forcleaning cane-juice during brown-sugar preparation (Shetty et al. 2013). Besides,it has been found to possess various pharmacological and medicinal propertiesagainst high-cholesterol, diabetes and cancer (Jenkins et al. 2005; Sabitha etal. 2011). Despite its economicalimportance, Bhendi production is massively affected by several abiotic andbiotic factors and yield losses due to biotic factors are quite significant(Jellis 2009).
The major biotic factors include various pathogens like fungi,bacteria, virus, mycoplasma, and nematode. The mostserious diseases caused by these pathogens are damping-off (Macrophominaphaseolina, Pythium aphanidermatum, and Rhizoctonia solani),vascular wilt (Fusarium oxysporum), Cercospora blight (Cercosporamalayensis), powdery mildew (Erysiphe cichoracearum, Oidiumabelmoschi), root and stem rot (Phytophthorapalmivora), rootknot nematode (Meloidogyne sp.), leaf spot (Alternaria sp.
, Cercospora malayensis), bhendi yellow vein mosaic disease(Bhendi yellow vein mosaic virus),and enation leaf curl disease (Enationleaf curl virus) (Abdel-Rehim et al. 1992, Tripathi, 1994; Sastry andSingh, 1974; Jose and Usha, 2003; Fajinmi and Fajinmi, 2006 & 2010; Singh,1996; Chandran et al. 2013; Sanwal et al.
2014). Amongthese, viruses are considered as a serious threat to bhendi production and itis observed that this crop is susceptible to at least 19 different plantviruses (Brunt et al. 1990, Swanson and Harrison 1993). Of these, bhendi yellow vein mosaic virus (BYVMV)and enation leaf curl virus ELCuV) belongs to the genus begomovirus (family:Geminiviridae) cause significant losses in bhendi production in terms of both yieldand quality. The infected plants are found to be associated with heavyinfestations of the whitefly Bemisiatabaci, the vector of begomoviruses. The loss in yield, due to YVMV and/or ELCuVin bhendi was found ranging from 30 to 100% depending on the age of the plantat the time of infection (Singh, 1996, Venkataravanappa etal.
2013). The descriptionof the family Geminiviridae to which bhendi yellow vein mosaic virus and enation leaf curl virus belongs aredetailed below:The family: GeminiviridaeThe family Geminiviridaeincludes plant-infecting viruses with circular ssDNA genomes that areencapsidated in a twinned icosahedral (or geminate) capsid (Zhang etal. 2001; Bottcher et al. 2004, Hanley-Bowdoin et al. 1999). Thisfamily contains more than 209 members of plant-infecting geminiviruses (Fauquetand Stanley, 2005). Geminiviruses are distributed globally and infect bothmonocotyledonous and dicotyledonous plants.
They are often responsible forserious yield losses in economically important crops, including cassava,chickpea, cotton, bhendi, tomato, maize and legumes (Moffat, 1999; Legg andFauquet, 2004; Briddon, 2003; Briddon and Markham, 2000; Jose and Usha, 2000; Morionesand Navas-castillo, 2000; Varma and Malathi, 2003). Symptoms of geminivirusinfections include yellowing, stunting, mosaic, curling, foliar crinkling, and/orstriations. Based on the genome organization, host range, insectvector, and sequence relationships, viruses in the family Geminiviridae are divided into nine genera, viz.
, Becurtovirus, Begomovirus,Capulovirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus,Topucovirus, and Turncurtovirus (Brown et al. 2012; Varsani et al. 2017). Thesegenera are named from the abbreviations of the type members Beet curlytop Iran virus (BCTIV, Becurtovirus),Bean golden mosaicvirus (BGMV, Begomovirus), Euphorbia caput-medusae latentvirus (EcmLV, Capulovirus), Beet curly topvirus (BCTV, Curtovirus), Eragrostiscurvula streak virus (ECSV, Eragrovirus),Grapevine red blotchvirus (GRBV, Grablovirus), Maizestreak virus (MSV, Mastrevirus),Tomato pseudo curlytop virus (TPCTV, Topocuvirus), and Turnip curly topvirus (TCTV, Turncurtovirus).
They are transmitted byleafhoppers (mastreviruses, becurtoviruses and curtoviruses), treehoppers(topocuviruses, grabuloviruses) and whiteflies (begomoviruses) (Buck, 1999;Jeske, 2009; ICTV 2017 (10th report)).Table 1.1: Geminiviruses, their type members and properties (Rojas etal. 2005; ICTV 2017 (http://www.ictvonline.
org/virusTaxonomy.asp)). Genus Genome organization Vector Host range Type member Becurtovirus Monopartite Leaf hopper Dicots Beet curly top Iran virus Capulovirus Monopartite Not yet determined Dicots Euphorbia capt-medusae latent virus Curtovirus Monopartite Leaf hopper Dicots Beet curly top virus Eragrovirus Monopartite Unknown Dicots Eragrostis curvula streak virus Grablovirus Monopartite Tree hopper Dicots Grapevine red blotch virus Mastrevirus Monopartite Leaf hopper Monocots and some dicots Maize streak virus Topocuvirus Monopartite Tree hopper Dicots Tomato pseudo curly top virus Turncurtovirus Monopartite Unknown Not yet determined Turnip curly top virus Begomovirus Monopartite or Bipartite Whiteflies Dicots Bean golden mosaic virus GenusBecurtovirusBecurtovirusesare transmitted by leafhoppers and infect dicots causing curly top disease. Thebiological characteristics of the becurtoviruses are similar to members of the genusCurtovirus. They have a monopartitegenome which encodes five proteins and genome organization resembles that of thegenus Mastrevirus (Sahu et al., 2013). GenusCapulovirusCapuloviruses have four proteins in thevirion sense strand and their arrangements are complex with respect to othergenus.
The ORF MP (movement protein) overlaps with the CP (coat protein) ORFand two or more MP ORFs constitute an intron containing MP. Capuloviruses have three proteins in theircomplementary sense strand in which, ORFs C1 and C2 overlaps with each otherand predicted to encode replication-associated protein (Rep) from a splicedtranscript. As similar to mastreviruses, it is possible that capuloviruses mayexpress a RepA protein from an unspliced transcript. The other ORF C3 completelylies within the ORF C1 (Fig. 1.1).
Capuloviruses have nonanucleotide motifTAATATTAC at their origin of replication (Bernardo et al. 2013). Four species: Alfalfa leaf curl virus, Euphorbiacapt-medusae latent virus, French bean severe leaf curl virus, Plantago lanceolatalatent virus are identifiedin this genus. Of these, Alfalfaleaf curl virus is shown to transmit by aphid (Roumagnac et al. 2015). Novector has been identified for the other three species in this genus.GenusCurtovirusCurtoviruses are transmitted byleafhoppers, infect dicotyledonous plants and have a monopartite genome(Stanley et al. 2005).
Curtoviruseshave a genome size of nearly 3 kb with an intergenic region that contains the originof replication and encodes seven proteins bidirectionally (Fig. 1.1). The three proteins V1 (capsid protein, CP), V2 (a single stranded (ss)/double stranded (ds) DNA regulator) and V3 (a putative movement protein, MP) inthe virion–sense and four proteins C1 (the replication associated protein,Rep), C2 (a pathogenicity- associated protein involved in a recovery phenotype),C3 (a replication enhancer protein, REn), and C4 (a protein affecting celldivision and symptom development) in the complementary-sense (Briddon et al.,1990). Beet culy topvirus (BCTV) belongs to this genus.
GenusEragrovirusEragroviruses are monopartite geminiviruses with a uniqueTAAGATTCC virion strand origin of replication and infect dicots. The genome of eragroviruses encodes two proteins in the virion sense V1 (Coat protein) and V2 (Movement protein) and two in the complementary sense, C1 (Replication initiation protein) and C2 (possible transcription activator protein). The insect vector is yet to be identified (Sahu et al., 2013). Genus GrablovirusGrabuloviruses are transmitted by treehoppers, have a monopartite genomewith 3.
2 kb which is significantly larger than those of other monopartitegeminiviruses (2.7-3.0 kb) (Bahder et al. 2016). The genome of grabulovirusesencodes three proteins in the virion sense V1 (Coat protein), V2 and V3(Movement protein) and three in the complementary sense, C1 and C2 (Replicationassociated protein), C3 protein function is unknown and is completely nested inthe ORF of C1 (Sudarshana et al.
2015; Krenz et al. 2014) (Fig. 1.1). Grapevine red blotch virus is a memberof this genus.
GenusMastrevirusMastreviruses are transmitted byleafhoppers, have a monopartite genome and mostly infect monocotyledonousspecies. The genus includes Maize streak virus (MSV) and Wheat dwarf virus (WDV). Some members Tobacco yellow dwarf virus (TYDV), and Bean yellow dwarf virus (BeYDV) infect dicotyledonous plants(Morris et al.
1992; Liu et al. 1998). There are two intergenic regions: the long intergenic region (LIR) andthe short intergenic region (SIR), required for completing the DNA replicationcycle. Mastrevirus genome encodes four proteins:RepA protein (exclusive to this genus) and Rep protein on the complementarysense strand and the movement protein (MP) and the coat protein (CP) on theviral sense strand (Palmer and Rybicki, 1998).GenusTopocuvirusThe topocuvirusesinfect dicots and aretransmitted by tree hoppers. They have monopartite genomewhich encodes six proteins. The coat protein has features of the leafhopper-transmitted mastreviruses whereas the organization of thecomplementary-sense genes is similar to that of the single-componentbegomoviruses. Therefore it might have arisen due tothe recombination between a curtoviruses andbegomoviruses (Briddon et al.
, 1996; Rojas et al, 2005). Tomato pseudo curly top virus belongs to this genus.Genus Turncurtovirus Turncurtovirus constitutes a single member (Turnipcurly top virus) with a monopartite genome which closely resembles membersof Curtovirus. The genome comprisesof six ORFs rather than seven ORFs: V1 (Coatprotein) and V2 (Movementprotein) in the virion sense and C1 (Replication initiation protein), C2 (Transcription activator protein), C3 (Replicationenhancer protein) and C4 (Symptom determinant) in the complementary sense. The insect vector is yet to be identified (Sahu et al.
, 2013). Genus BegomovirusBegomovirus is the largest genus of family Geminiviridaeand is transmitted by the whitefly Bemicia tabaci and infectsdicotyledonous plants. The name of the genus Begomovirus is derived from the firsttwo letters of the each word of the type species, Bean golden mosaic virus (BGMV) causing golden mosaic disease in bean in CentralAmerica. Begomoviruses are transmitted by only one vector species, whitefly (Bemisia tabaci). About 68.
1% of geminiviruses belong to the genus Begomovirus. Presently,322 virus species have beenofficially recognised under the genus Begomovirus, which is the maximumnumberof members so far known in any genera of plant viruses. Symptoms of begomoviruses infected plants areyellow mosaic, leaf distortion, curling and stunting. Infection in earlyseedling stage leads to poor fruit set and infertile seeds, resulting in severeyield loss. Begomoviruses infect a large number of dicots such as bhendi,cassava, cotton, legumes, tomato, chilli, and many more. Some of the diseaseslike bhendi yellow vein mosaic, okra enation leaf curl, cassava mosaic andcotton leaf curl are known for century and cause huge economic loss (Varma andMalathi, 2003). Genus Begomovirus consists of bothbipartite and monopartite members. Based on the geographical distributionbegomoviruses are classified as “Old world” (Eurasia, Africa and Australasia)or “New World” (America).
Most ofthe “Old World” and all the “New World” begomoviruses have bipartite genomeswhereas some of the “Old World” begomoviruses have monopartite genomes. Thegenome of bipartite begomoviruses consists of two genomic components,designated DNA A and DNA B which arecovalently closed, circular, 2.5- to 2.9-kb-long single-stranded (ss) DNAmolecules (Lazarowitz, 1992). DNA A depends on DNA B for intracellular andintercellular movement.
DNA B depends on DNA A for replication andencapsidation (Rogers et al., 1986;Sunter et al., 1987; Townsend et al.
, 1986). Inorder to facilitate recognition of DNA B by the Rep encoded by DNA A. a segmentof 110-200 nt sequences is present within the intergenic region and are highlyconserved between DNA A and DNA B component.
This region is denoted as commonregion (CR) and is highly identical between DNA A and DNA B component. Theintergenic region consists of repetitive elements called iterons upstream ofthe highly conserved stem-loop structure. The iterons, represent the bindingsites of Rep. The invariant nonanucleotide sequence TAA TAT TAC present in theloop is conserved in all geminiviruses and the nicking between seventh andeight nucleotide by Rep protein is proved to initiate replication (Harrisonand Robinson, 1999; Hanley-Bowdoin et al. 1999, 2013; Guiterrez, 2000).
Thebipartite begomoviruses have five or six ORFs on DNA A, one or two on thevirion-sense strand and four or five in the complementary sense strand. The DNAB component encodes two ORFs one each in the complementary and virion senserespectively. In DNA A, AC1 encodes Rep which is involved in thereplication of the virus genome, AC2 encodes a transcription activator protein(TrAP) which activates transcription from AV1, BC1 and BV1 promoters(Sunter and Bisaro, 1991; Shivaprasad etal., 2005), AC3 encodes aReplication enhancer (REn)protein that increases the efficiency of viral replication (Settlage et al., 2005) and AC4 protein is believed to be involved incontrolling the cell cycle progression and is a symptom determinant and suppressor ofpost-transcriptional gene silencing (Bisaro, 2006; Vanitharani et al.
, 2004). AV1encodes the coat protein, and AV2 encodes the pre-coat protein. AV2 is notpresent in “New World” begomoviruses. BV1encodes the Nuclear shuttle protein (NSP) and BC1encodes the Movement protein (MP) involved in cell to cell movement (Rojas etal., 2005). Tomato golden mosaic virus (TGMV), SriLankan cassava mosaic virus (SLCMV), Mungbean yellow mosaic virus (MYMV), African cassava mosaic virus (ACMV)belong to this genus.
Satellite DNA associated withgeminivirusesSatellites are defined as viruses ornucleic acids that depend on a helper virus for their replication andencapsidation but lack extensive nucleotide sequence homology to the helpervirus and are dispensable for its proliferation (Murant and Mayo 1982; Mayo etal., 2005). The monopartite begomoviruses in general and few of thebipartite begomoviruses of the old world are associated with additionalcircular ssDNA components referred as satellites. There are three types of satellites identifiedtill now and they are aphasatellite, betasatellite, and deltasatellite. Thefirst begomovirus satellite to be discovered, ToLCV-sat, was isolated fromtomato plants infected with the monopartite begomovirus Tomato leaf curl virus (ToLCV) in Australia (Dry et al., 1997). The circular satellite issmall (682 nucleotides) without any open reading frames and has little sequencesimilarity to its helper virus, with the exception of the ubiquitousgeminivirus TAATATTAC motif and ToLCV Rep binding motif, which are present(Behjatnia et al., 1998).
ToLCV-satis not essential for ToLCV infectivity and does not have any effect on thesymptoms induced by the helper virus, but is dependent on the helperbegomovirus for its replication and encapsidation and hence has the hallmarksof a satellite DNA.AlphasatelliteBegomovirus-associated alphasatellites(previously called DNA1) are self-replicating satellite-like molecules,dependent on the helper virus for movement, encapsidation and vectortransmission. They areapproximately half the size of its helper begomovirus (1370 nucleotides) andencodes Rep. Alphasatellites have been found in association with severaldistinct monopartite begomoviruses that have been isolated from a range ofplant species growing in different regions throughout Africa and Asia (Mansoor et al.
, 1999; 2000a; 2000b; 2001;Saunders and Stanley, 1999; Briddon et al.,2004; Wu and Zhou, 2005, Chandran et al. 2013). Alphasatelliteshave a highly conserved genome organization, encompassing areplication-associated protein of nearly 36 kDa, an adenine-rich region ofnearly 200 nts and an origin of replication (Ori) (including a conservednona-nucleotide TAGTATT/AC), similar to nanoviruses. There is no known specificfunction attributed to alphasatellites. Recently, Nawaz-ul-Rehman et al.
, (2010) haveshown that alpha Rep can function as PTGS suppressor and also interact with Repand C4 of the helper begomovirus.BetasatelliteA novelssDNA component named DNA b was isolated and shown to induce the yellow vein phenotype when inoculatedalong with AYVV into ageratum (Saunders etal., 2000).
DNA b associated with many monopartite begomoviruses is shown to infect adiverse range of plants including bhendi, hibiscus, hollyhock, Mallow (Malvaceae), honeysuckle (Caprifoliaceae), tomato, tobacco, andchilli (Solanaceae), squash (Cucurbitaceae), zinnia and ageratum (Asteraceae) (Briddon et al. 2003; Zhouet al. 2003; Jose and Usha, 2000; Venkataravanappa et al. 2011, 2012a, 2012b, 2013a, 2013b). DNA b completely depends on the helper begomovirusfor its replication and encapsidation. DNAb is approximately half the size (1300-1400 nucleotides) of theirhelper begomovirus. DNA b shares no sequence homology with the helpercomponents except a similar nona-nucleotide (TAATATT/AC).
Although some levelof specificity exists for trans-replication of betasatellites, they generallyhave a loose specificity of trans-replication by different helper components. For example, the ToLCV satellite, which isbelieved to be a defective b component can replicate and spreadsystemically in association with distinct begomoviruses such as Tomato yellow leaf curl virus (TYLCV), African cassava mosaic virus (ACMV) andBCTV (Dry et al., 1997). Similarly AYVD b component canalso be maintained in plants by Sri Lankan cassava mosaic virus (SLCMV),indicating that they are promiscuous and hence have the potential to exchangehelper viruses during mixed infections (Saunders et al.
, 2002). bC1has been shown to play major roles in pathogenicity, symptom modulation, viralmovement and suppression of post transcriptional gene silencing (PTGS)(Saunders et al., 2000, 2004; Briddonet al., 2003; Mansoor et al.
, 2003; Cui et al., 2004; Cui et al.,2005a,b; Saeed et al., 2005; Briddonand Stanley, 2006; Gopal et al.,2007).bC1proteinComparison of various DNA ? sequences revealed that the position andsize of the ?C1 ORF is conserved (Briddon etal. 2003). bC1 has been shown to play major roles in pathogenicity, symptommodulation, viral movement or suppression of post transcriptional gene silencing(PTGS) (Saunders et al.
, 2000; 2004,Briddon et al., 2003; Mansoor et al., 2003; Saeed et al., 2005; Cui et al.,2004;Cui et al., 2005a; 2005b;Briddon and Stanley, 2006; Gopal et al.
,2007). Integration of DNA-b or ORF bC1 into the N. benthamiana or N.tabacum genome resulted in severe developmentalabnormalities in transgenic plants, indicating that the component encodes apathogenicity determinant that is functional in the absence of the helperbegomovirus (Saunders et al., 2004;Cui et al., 2005a; Saeed et al., 2005; Kumar et al.
, 2006; Gopal et al.,2007). A single gene (bC1) on the complementary-sense strand of AYVD DNA-b with the potential to encode a protein ofapproximately 14 kDa has been identified by transcript mapping (Saunders et al., 2004) and a similar geneticorganization has been shown for Cotton leaf curl disease (CLCuD) DNA-b (Saeed etal., 2005). Analysis of naturally occurring CLCuD DNA-b mutants lacking entire bC1 coding region attributed bC1 to the pathogenicity of DNA-b (Briddon etal., 2003; Tao and Zhou, 2004). These mutants were trans-replicated by thehelper virus and moved systemically throughout the plant but were unable toinduce typical disease symptoms.
It has also been suggested that bC1 may be involved in the movement of thevirus (Briddon and Stanley, 2006). CLCuDbC1 can help in the movement of the bipartite begomovirus Tomatoleaf curl New Delhi virus DNA-A component in the absence of DNA-B, as wellas mutants of V1 and C4 (which are normally implicated in virus movement)encoded by the monopartite begomovirus ToLCNDV suggesting the involvement of bC1 in virus movement. The bC1 encoded by the DNA-b component associated with Tomato yellowleaf curl China virus (TYLCCNV) has been shown to suppresspost-transcriptional gene silencing. It was shown that the suppressor activityas well as symptom induction requires the nuclear localization of bC1 (Cui etal.
, 2005b). The bC1 has been shown to bind to both ssDNA and dsDNA in a sequencenon-specific manner (Briddon and Stanley, 2006).DeltasatelliteA novel non-coding sub viral molecule namedas deltasatellites (Lazano et al. 2016) have been identified recently withbegomoviruses infecting sweet potato (sweepoviruses).
They are structurallysimilar to subgenomic betasatellite associated with tomato leaf curl virus (ToLCV) from Australia; they have theconserved stem and loop structure with nonanucleotide sequence TAATATAC and SCRsimilar to betasatellites. The contribution of deltasatellites to viralpathogenicity is yet to be understood.