Introduction hectare land (FAOSTAT, 2014). Bhendi is a multipurpose

Introduction

Abelmoschus
esculentus (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 its
caducous nature of the calyx (Dhankhar et al., 2005). The word ‘Abelmoschus’
is originated from the Arabian word “abul-l-mosk” meaning “source of
musk,” denoting to the musky smell of the
seeds.  Bhendi is widely cultivated
in the temperature regions of Asia, Africa and
Southern Europe (Charrier, 1984).  This
plant is thought to be the native of South Africa and the first recorded
reference was from Egyptians in 1216 A.D (Lamont, 1999). However, ten species of bhendi plants are present in India, of which A. esculentus
is the only cultivated species (Dhankar et al. 2005). Bhendi is an allopolyploid with the lowest chromosome
number in A. angulus (2n=56) and the
highest in A. caillei (2n=100), which
is an amphiployploid between A. esculentus (2n=13-140) and A. manihot
(2n=60-68) (Siemonsma, 1982). Developing countries like Asia and Africa
contribute more than 99% of okra cultivation compared with other parts of the
world. Globally, bhendi cultivation is occupying an area of 1.83 million with
the annual yield of 9.62 million metric tons (MT). India contributes first in
the world with 6.5 million metric tons (72% of the total world production) of bhendi
produced from over 0.5 million hectare land (FAOSTAT, 2014).

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            Bhendi is a multipurpose crop with
huge 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 rich
source of oil (30-40%) and proteins (15-20%) (Gemede et al. 2015). Its mature
seed and stems are ample source of crude fibre and used in paper industry (Kochlar, 1986). The roots and stems are used for
cleaning cane-juice during brown-sugar preparation (Shetty et al. 2013). Besides,
it has been found to possess various pharmacological and medicinal properties
against high-cholesterol, diabetes and cancer (Jenkins et al. 2005; Sabitha et
al. 2011).

            Despite its economical
importance, Bhendi production is massively affected by several abiotic and
biotic 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 most
serious diseases caused by these pathogens are damping-off (Macrophomina
phaseolina, Pythium aphanidermatum, and Rhizoctonia solani),
vascular wilt (Fusarium oxysporum), Cercospora blight (Cercospora
malayensis), powdery mildew (Erysiphe cichoracearum, Oidium
abelmoschi), root and stem rot (Phytophthora
palmivora), root
knot nematode (Meloidogyne sp.), leaf spot (Alternaria sp., Cercospora malayensis), bhendi yellow vein mosaic disease
(Bhendi yellow vein mosaic virus),
and enation leaf curl disease (Enation
leaf curl virus) (Abdel-Rehim et al. 1992, Tripathi, 1994; Sastry and
Singh, 1974; Jose and Usha, 2003; Fajinmi and Fajinmi, 2006 & 2010; Singh,
1996; Chandran et al. 2013; Sanwal et al. 2014). Among
these, viruses are considered as a serious threat to bhendi production and it
is observed that this crop is susceptible to at least 19 different plant
viruses (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 yield
and quality. The infected plants are found to be associated with heavy
infestations of the whitefly Bemisia
tabaci, the vector of begomoviruses. The loss in yield, due to YVMV and/or ELCuV
in bhendi was found ranging from 30 to 100% depending on the age of the plant
at the time of infection (Singh, 1996, Venkataravanappa et
al. 2013).

            The description
of the family Geminiviridae to which bhendi yellow vein mosaic virus and enation leaf curl virus belongs are
detailed below:

The family: Geminiviridae

The family Geminiviridae
includes plant-infecting viruses with circular ssDNA genomes that are
encapsidated in a twinned icosahedral (or geminate) capsid (Zhang et
al. 2001; Bottcher et al. 2004, Hanley-Bowdoin et al. 1999). This
family contains more than 209 members of plant-infecting geminiviruses (Fauquet
and Stanley, 2005). Geminiviruses are distributed globally and infect both
monocotyledonous and dicotyledonous plants. They are often responsible for
serious yield losses in economically important crops, including cassava,
chickpea, cotton, bhendi, tomato, maize and legumes (Moffat, 1999; Legg and
Fauquet, 2004; Briddon, 2003; Briddon and Markham, 2000; Jose and Usha, 2000; Moriones
and Navas-castillo, 2000; Varma and Malathi, 2003). Symptoms of geminivirus
infections include yellowing, stunting, mosaic, curling, foliar crinkling, and/or
striations. Based on the genome organization, host range, insect
vector, 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).  These
genera are named from the abbreviations of the type members Beet curly
top Iran virus (BCTIV, Becurtovirus),
Bean golden mosaic
virus (BGMV, Begomovirus), Euphorbia caput-medusae latent
virus (EcmLV, Capulovirus), Beet curly top
virus (BCTV, Curtovirus), Eragrostis
curvula streak virus (ECSV, Eragrovirus),
Grapevine red blotch
virus (GRBV, Grablovirus),
 Maize
streak virus (MSV, Mastrevirus),
Tomato pseudo curly
top virus (TPCTV, Topocuvirus), and Turnip curly top
virus (TCTV, Turncurtovirus). They are transmitted by
leafhoppers (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 et
al. 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

Genus
Becurtovirus

Becurtoviruses
are transmitted by leafhoppers and infect dicots causing curly top disease. The
biological characteristics of the becurtoviruses are similar to members of the genus
Curtovirus. They have a monopartite
genome which encodes five proteins and genome organization resembles that of the
genus Mastrevirus (Sahu et al., 2013).

Genus
Capulovirus

Capuloviruses have four proteins in the
virion sense strand and their arrangements are complex with respect to other
genus. The ORF MP (movement protein) overlaps with the CP (coat protein) ORF
and two or more MP ORFs constitute an intron containing MP.  Capuloviruses have three proteins in their
complementary sense strand in which, ORFs C1 and C2 overlaps with each other
and predicted to encode replication-associated protein (Rep) from a spliced
transcript. As similar to mastreviruses, it is possible that capuloviruses may
express a RepA protein from an unspliced transcript. The other ORF C3 completely
lies within the ORF C1 (Fig. 1.1). Capuloviruses have nonanucleotide motif
TAATATTAC at their origin of replication (Bernardo et al. 2013). Four species: Alfalfa leaf curl virus, Euphorbia
capt-medusae latent virus, French bean severe leaf curl virus, Plantago lanceolata
latent virus are identified
in this genus. Of these, Alfalfa
leaf curl virus is shown to transmit by aphid (Roumagnac et al. 2015). No
vector has been identified for the other three species in this genus.

Genus
Curtovirus

Curtoviruses are transmitted by
leafhoppers, infect dicotyledonous plants and have a monopartite genome
(Stanley et al. 2005). Curtoviruses
have a genome size of nearly 3 kb with an intergenic region that contains the origin
of 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) in
the 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 cell
division and symptom development) in the complementary-sense (Briddon et al.,
1990). Beet culy top
virus (BCTV) belongs to this genus.

Genus
Eragrovirus

Eragroviruses are monopartite geminiviruses with a unique
TAAGATTCC 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 Grablovirus

Grabuloviruses are transmitted by treehoppers, have a monopartite genome
with 3.2 kb which is significantly larger than those of other monopartite
geminiviruses (2.7-3.0 kb) (Bahder et al. 2016). The genome of grabuloviruses
encodes three proteins in the virion sense V1 (Coat protein), V2 and V3
(Movement protein) and three in the complementary sense, C1 and C2 (Replication
associated protein), C3 protein function is unknown and is completely nested in
the ORF of C1 (Sudarshana et al. 2015; Krenz et al. 2014) (Fig. 1.1). Grapevine red blotch virus is a member
of this genus.

Genus
Mastrevirus

Mastreviruses are transmitted by
leafhoppers, have a monopartite genome and mostly infect monocotyledonous
species.  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) and
the short intergenic region (SIR), required for completing the DNA replication
cycle.  Mastrevirus genome encodes four proteins:
RepA protein (exclusive to this genus) and Rep protein on the complementary
sense strand and the movement protein (MP) and the coat protein (CP) on the
viral sense strand (Palmer and Rybicki, 1998).

Genus
Topocuvirus

The topocuviruses
infect dicots and are
transmitted by tree hoppers. They have monopartite genome
which encodes six proteins.  The coat protein has features of the leaf
hopper-transmitted mastreviruses whereas the organization of the
complementary-sense genes is similar to that of the single-component
begomoviruses. Therefore it might have arisen due to
the recombination between a curtoviruses and
begomoviruses (Briddon et al., 1996; Rojas et al, 2005). Tomato pseudo curly top virus belongs to this genus.

Genus Turncurtovirus

Turncurtovirus constitutes a single member (Turnip
curly top virus) with a monopartite genome which closely resembles members
of Curtovirus. The genome comprises
of six ORFs rather than seven ORFs: V1 (Coat
protein) and V2 (Movement
protein) in the virion sense and C1 (Replication initiation protein), C2 (Transcription activator protein), C3 (Replication
enhancer protein) and C4 (Symptom determinant) in the complementary sense. The insect vector is yet to be identified (Sahu et al., 2013).    

 

 Genus Begomovirus

Begomovirus is the largest genus of family Geminiviridae
and is transmitted by the whitefly Bemicia tabaci and infects
dicotyledonous plants. The name of the genus Begomovirus is derived from the first
two letters of the each word of the type species, Bean golden mosaic virus (BGMV) causing golden mosaic disease in bean in Central
America. 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 been
officially recognised under the genus Begomovirus, which is the maximum
number
of members so far known in any genera of plant viruses.  Symptoms of begomoviruses infected plants are
yellow mosaic, leaf distortion, curling and stunting. Infection in early
seedling stage leads to poor fruit set and infertile seeds, resulting in severe
yield loss. Begomoviruses infect a large number of dicots such as bhendi,
cassava, cotton, legumes, tomato, chilli, and many more. Some of the diseases
like bhendi yellow vein mosaic, okra enation leaf curl, cassava mosaic and
cotton leaf curl are known for century and cause huge economic loss (Varma and
Malathi, 2003).

            Genus Begomovirus consists of both
bipartite and monopartite members. Based on the geographical distribution
begomoviruses are classified as “Old world” (Eurasia, Africa and Australasia)
or “New World” (America). Most of
the “Old World” and all the “New World” begomoviruses have bipartite genomes
whereas some of the “Old World” begomoviruses have monopartite genomes. The
genome of bipartite begomoviruses consists of two genomic components,
designated DNA A and DNA B  which are
covalently closed, circular, 2.5- to 2.9-kb-long single-stranded (ss) DNA
molecules (Lazarowitz, 1992).  DNA A depends on DNA B for intracellular and
intercellular movement. DNA B depends on DNA A for replication and
encapsidation (Rogers et al., 1986;
Sunter et al., 1987; Townsend et al., 1986). In
order to facilitate recognition of DNA B by the Rep encoded by DNA A. a segment
of 110-200 nt sequences is present within the intergenic region and are highly
conserved between DNA A and DNA B component. This region is denoted as common
region (CR) and is highly identical between DNA A and DNA B component. The
intergenic region consists of repetitive elements called iterons upstream of
the highly conserved stem-loop structure. The iterons, represent the binding
sites of Rep. The invariant nonanucleotide sequence TAA TAT TAC present in the
loop is conserved in all geminiviruses and the nicking between seventh and
eight nucleotide by Rep protein is proved to initiate replication (Harrison
and Robinson, 1999; Hanley-Bowdoin et al. 1999, 2013; Guiterrez, 2000). The
bipartite begomoviruses have five or six ORFs on DNA A, one or two on the
virion-sense strand and four or five in the complementary sense strand. The DNA
B component encodes two ORFs one each in the complementary and virion sense
respectively.  In DNA A, AC1 encodes Rep which is involved in the
replication 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 et
al., 2005), AC3 encodes a
Replication enhancer (REn)
protein that increases the efficiency of viral replication (Settlage et al., 2005) and AC4 protein is believed to be involved in
controlling the cell cycle progression and is a  symptom determinant and suppressor of
post-transcriptional gene silencing (Bisaro, 2006; Vanitharani et al., 2004).  AV1
encodes the coat protein, and AV2 encodes the pre-coat protein. AV2 is not
present in “New World” begomoviruses.  BV1
encodes the Nuclear shuttle protein (NSP) and BC1
encodes the Movement protein (MP) involved in cell to cell movement (Rojas et
al., 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 with
geminiviruses

Satellites are defined as viruses or
nucleic acids that depend on a helper virus for their replication and
encapsidation but lack extensive nucleotide sequence homology to the helper
virus and are dispensable for its proliferation (Murant and Mayo 1982; Mayo et
al., 2005). The monopartite begomoviruses in general and few of the
bipartite begomoviruses of the old world are associated with additional
circular ssDNA components referred as satellites.  There are three types of satellites identified
till now and they are aphasatellite, betasatellite, and deltasatellite. The
first begomovirus satellite to be discovered, ToLCV-sat, was isolated from
tomato plants infected with the monopartite begomovirus Tomato leaf curl virus (ToLCV) in Australia (Dry et al., 1997). The circular satellite is
small (682 nucleotides) without any open reading frames and has little sequence
similarity to its helper virus, with the exception of the ubiquitous
geminivirus TAATATTAC motif and ToLCV Rep binding motif, which are present
(Behjatnia et al., 1998). ToLCV-sat
is not essential for ToLCV infectivity and does not have any effect on the
symptoms induced by the helper virus, but is dependent on the helper
begomovirus for its replication and encapsidation and hence has the hallmarks
of a satellite DNA.

Alphasatellite

Begomovirus-associated alphasatellites
(previously called DNA1) are self-replicating satellite-like molecules,
dependent on the helper virus for movement, encapsidation and vector
transmission. They are
approximately half the size of its helper begomovirus (1370 nucleotides) and
encodes Rep. Alphasatellites have been found in association with several
distinct monopartite begomoviruses that have been isolated from a range of
plant 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). Alphasatellites
have a highly conserved genome organization, encompassing a
replication-associated protein of nearly 36 kDa, an adenine-rich region of
nearly 200 nts and an origin of replication (Ori) (including a conserved
nona-nucleotide TAGTATT/AC), similar to nanoviruses. There is no known specific
function attributed to alphasatellites. Recently, Nawaz-ul-Rehman et al., (2010) have
shown that alpha Rep can function as PTGS suppressor and also interact with Rep
and C4 of the helper begomovirus.

Betasatellite

A novel
ssDNA component named DNA b was isolated and shown to induce the yellow vein phenotype when inoculated
along with AYVV into ageratum (Saunders et
al., 2000). DNA b associated with many monopartite begomoviruses is shown to infect a
diverse range of plants including bhendi, hibiscus, hollyhock, Mallow (Malvaceae), honeysuckle (Caprifoliaceae), tomato, tobacco, and
chilli (Solanaceae), squash (Cucurbitaceae), zinnia and ageratum (Asteraceae) (Briddon et al. 2003; Zhou
et al. 2003; Jose and Usha, 2000; Venkataravanappa et al. 2011, 2012a, 2012b, 2013a, 2013b). DNA b completely depends on the helper begomovirus
for its replication and encapsidation.  DNA
b is approximately half the size (1300-1400 nucleotides) of their
helper begomovirus. DNA b shares no sequence homology with the helper
components except a similar nona-nucleotide (TAATATT/AC). Although some level
of specificity exists for trans-replication of betasatellites, they generally
have a loose specificity of trans-replication by different helper components.  For example, the ToLCV satellite, which is
believed to be a defective b component can replicate and spread
systemically in association with distinct begomoviruses such as Tomato yellow leaf curl virus (TYLCV), African cassava mosaic virus (ACMV) and
BCTV (Dry et al., 1997). Similarly AYVD b component can
also be maintained in plants by Sri Lankan cassava mosaic virus (SLCMV),
indicating that they are promiscuous and hence have the potential to exchange
helper viruses during mixed infections (Saunders et al., 2002). bC1
has been shown to play major roles in pathogenicity, symptom modulation, viral
movement and suppression of post transcriptional gene silencing (PTGS)
(Saunders et al., 2000, 2004; Briddon
et al., 2003; Mansoor et al., 2003; Cui et al., 2004; Cui et al.,
2005a,b; Saeed et al., 2005; Briddon
and Stanley, 2006; Gopal et al.,
2007).

bC1
protein

Comparison of various DNA ? sequences revealed that the position and
size of the ?C1 ORF is conserved (Briddon et
al. 2003). bC1 has been shown to play major roles in pathogenicity, symptom
modulation, 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 developmental
abnormalities in transgenic plants, indicating that the component encodes a
pathogenicity determinant that is functional in the absence of the helper
begomovirus (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 of
approximately 14 kDa has been identified by transcript mapping (Saunders et al., 2004) and a similar genetic
organization has been shown for Cotton leaf curl disease (CLCuD) DNA-b (Saeed et
al., 2005). Analysis of naturally occurring CLCuD DNA-b mutants lacking entire bC1 coding region attributed bC1 to the pathogenicity of DNA-b (Briddon et
al., 2003; Tao and Zhou, 2004). These mutants were trans-replicated by the
helper virus and moved systemically throughout the plant but were unable to
induce typical disease symptoms. It has also been suggested that bC1 may be involved in the movement of the
virus (Briddon and Stanley, 2006).  CLCuD
bC1 can help in the movement of the bipartite begomovirus Tomato
leaf curl New Delhi virus DNA-A component in the absence of DNA-B, as well
as 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 yellow
leaf curl China virus (TYLCCNV) has been shown to suppress
post-transcriptional gene silencing. It was shown that the suppressor activity
as well as symptom induction requires the nuclear localization of bC1 (Cui et
al., 2005b). The bC1 has been shown to bind to both ssDNA and dsDNA in a sequence
non-specific manner (Briddon and Stanley, 2006).

Deltasatellite

A novel non-coding sub viral molecule named
as deltasatellites (Lazano et al. 2016) have been identified recently with
begomoviruses infecting sweet potato (sweepoviruses). They are structurally
similar to subgenomic betasatellite associated with tomato leaf curl virus (ToLCV) from Australia; they have the
conserved stem and loop structure with nonanucleotide sequence TAATATAC and SCR
similar to betasatellites. The contribution of deltasatellites to viral
pathogenicity is yet to be understood.