2. review of literature - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/30692/9/09...2: review...
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2 REVIEW OF LITERATURE
2.1. Virus Diseases of Cucurbits
Cucurbits represent a widely diverse group of annual and perennial herbaceous
plants in over twelve families. Several of these species are host of many
pathogens that have developed pathogenic specificity for related plants
belonging to one or more families. The diseases caused by viruses are
particularly important as they affect agriculture production, food security and
income, particularly in the tropical parts of the world (Raj et al., 2007b).
A list of viruses causing epidemics on cucurbits in developing
countries is listed in Table 2.1:
Table 2.1. Viruses causing local epidemics in cucurbits in developing
country.
Virus Means of spread
Distribution in developing Country
1. Begomovirus Melon chlorotic leaf curl virus White flies Gautemala Squash leaf curl virus White flies China Squash yellow mottle virus White flies Costa Rica Tomato leaf curl virus White flies India, Pakistan,
Thailand 2. Comovirus Squash mosaic virus Beetle, seeds World wide 3. Ipomovirus Cucumber vein yellowing virus White flies Middle East
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4. Potyvirus Melon vein banding mosaic virus Aphids Taiwan Telfairia mosaic virus Aphids, seeds Nigeria Zucchini yellow fleck virus Aphids Mediterrarean 5. Tobamovirus Cucumber green mottle mosaic virus Contact Asia 6. Tospovirus Watermelon bud necrosis virus Thrips India Watermelon silver mottle virus Thrips Taiwan Zucchini lethal chlorosis virus Thrips Brazil
The importance of plant viruses in relation to crop production can be
realized from the fact that among the various factors responsible for low
yields, viral diseases are prominent and cause losses in world's crop
production amounting to several million rupees, which comes next only to
losses caused by insect pests. Plant virus diseases may damage leaves, stems,
roots, fruits, seed or flowers and may cause economic losses by reduction in
yield and quality of plant products. However, it is quite difficult to present
accurate estimates of the losses due to viral diseases.
Leaf curl diseases (LCDs) are caused by a number of viruses of the
genus Begomovirus—bean golden mosaic virus—(family Geminiviridae) that
collectively threaten vegetable and particularly cucumber production
throughout the tropical and subtropical regions of the world (Moriones and
Navas-Zastillo, 2000). In 1950s-1970s, Begomovirus disease in cucurbits was
of minor importance in India. During this time, only pumpkin (Cucurbita
moschata) yellow vein mosaic (PYVM) disease was known to occur in
central-western India (Varma, 1955). It attracted the attention as emerging
disease problems since 1981, when many other cucurbits such as bitter gourd,
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cucumber, muskmelon, and sponge gourd, winter squash were observed to be
affected by Begomovirus diseases (Raj and Singh, 1996; Varma and Giri,
1998; Singh et al., 2001; Khan et al., 2002; Muniyappa et al., 2003; Sohrab et
al., 2003; Rajnimala and Rabindran, 2007; Rajnimala et al., 2009;
Heydarnejad et al., 2009; Kumar et al., 2009).
Many different Begomovirus infecting cucurbits have been reported
from other countries such as Squash leaf curl Yunnan virus from China (Xie
and Zhou, 2003), Melon chlorotic leaf curl virus from Guatemala (Brown et
al., 2001), ToLCNDV-(Luf) from Thailand (Samertwanich et al., 2000),
Watermelon chlorotic stunt virus from Sudan and Iran (Kheyr-Pour et al.,
2000), Squash leaf curl Philippines virus from the Philippines (Kon et al.,
2003), Cucurbit leaf curl virus (Brown, 2000), Cucurbit leaf crumple virus
(Guzman et al., 2000), Squash leaf curl virus (Flock and Mayhew, 1981;
Lazarowitz and Lazdins, 1991) and Squash mild leaf curl virus (Brown et al.,
2002) from USA, and Luffa yellow mosaic virus from Vietnam (Revill et al.,
2003). Viruses affecting cultivation of cucurbitaceous crops in India and
abroad are as follows:
2.2. Major Cucurbit Viruses
The different viruses occurring on different species of cucurbits are briefly
described as herein:
2.2.1. Cucumber mosaic virus
Cucumber mosaic virus (CMV) is a type member of genus Cucumovirus,
family Bromoviridae. CMV is economically important because of its
capability to infect a large number of plant species and has widest host range
among all known plant viruses. CMV exists in numerous strains that differ in
their hosts, symptoms, ways of their transmission and in some other properties
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and characteristics. The concentration of the virus increases for several days
following inoculation, then decreases until the plant dies (Francki et al., 1979).
Cucumoviruses have icosahedra particles of 29 nm in diameter with
180 capsid protein subunits. The molecular weight of CMV falls in the range
of 5.8 to 6.7 million which consists of about 18% RNA and the remaining 82
per cent protein (Francki and Hatta, 1980). The RNA is tightly packed by the
protein shell, leaving a hollow core of about 110Ao along the threshold axes
(Smith et al., 2000).
CMV causes a variety of symptoms and different degree of losses to
various economically important vegetable food crops. CMV seldom attacks
the seedlings of cucurbits and causes mosaic. The severe mosaic was observed
in case of Luffa cylindrica. Later, infected leaves become mottled, distorted
and wrinkled. The subsequent growth of the infected plants is reduced and
they appear as dwarfed: shorter stem internodes and petioles, and under
developed leaves. Infected plants produce few runners and also few flowers
and fruit. Older leaves develop chlorotic and then necrotic areas along the
margins which later spread over the entire leaf. Cucumbers produced after
infections have pale green or white area intermingled with dark green, bumpy
areas. Fruits produced by the plants in the later stages of the disease are
somewhat miss-shape but have a smooth grow-white color with some irregular
green areas, these are often called “white pickle”. Cucumbers with cucumber
mosaic infection may have a bitter taste and make soggy pickles (Raj et al.,
2007b).
CMV is one of the most widespread plant viruses in the world
(Rossinck et al., 1999). The infection of this virus is not limited to the crop
plant only; weed-hosts also function as a reservoir for the virus and serve as
sources of inoculums for the development of disease epidemics in nearby crop
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lands. Transmission through planting materials is also significant in some crop
and weed hosts (Hsu et al., 2000). CMV has a wide range of hosts and attack a
great variety of vegetables, ornamentals, and other plants comprising as many
as 1191 host species in 40 families (Palukaities et al., 1992).
Cucurbita pepo is one of the largest consumable food crops in Asia.
Studies on mosaic disease of C. pepo have confirmed the presence of CMV
(Reddy and Narriani, 1963). CMV infected plants showed typical light and
deep green mosaic patterns on the affected leaves and slight reduction in leaf
size. Flowering is delayed when plants are infected at early stage of growth.
Fruits are reduced in size, paler in colour and occasionally chlorotic spots are
seen on the outer skin of young fruits. Incidence of the disease in the field was
46.5%.
Virus is sap transmissible and by Aphis gossypii Glov., A. craccivora
Kotch, A. evonymi Fabr. and Myzus persicae Sulz. Virus is also seed
transmitted, but to a very limited extent. Host range of the virus includes:
Nicotiana tabacum, N. glutinosa, Cucumis sativas, C. melo, C. anguria,
Cucurbita moschata, C. pepo, C. maxima, Lagenaria sicerarea, Trichosanthes
anguina, Momordica charantia, Luffa acutangula, Luffa cylindrica, Citrullus
vulgaris, Zinnia elegans, Gomphrena globosa, Brassica campestris and
Hesperis matronalis. These are, however, symptomless carriers (Chupp and
Sherf, 1960; Huang et al., 1987; Chabbouh and Cherif, 1990).
The genome of Cucumoviruses consists of three single stranded
messenger sense RNAs designated as 1, 2 and 3 in order of decreasing size.
RNAs 1 and 2 are encapsulated separated whereas RNA 3 and sub genomic
RNA 4 are probably encapsidated in the same particle (Lot and Kaper, 1976).
RNA 1 encodes one open reading frame, whereas RNA 2 and 3 each encodes
two open reading frames. RNA 1 and RNA 2 encode the proteins 1a and 2a,
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respectively, essential for replication (Hayes and Buck, 1990). RNA 2 also
codes for a second protein called 2b that affects the long distance movement
and symptomatology of the virus (Ding et al., 1995) RNA 3 is dicistronic and
encodes two proteins, Movement protein (MP) and Coat protein (CP). MP
encoded by an ORF located within the 5’-half of RNA 3 is involved in cell-to-
cell movement of the virus (Canto et al., 1997; Suzuki et al., 1991). The
second ORF located within the 3’-half encodes the 24 KDa CP, which is
expressed through a sub-genomic RNA 4 (Palukaitis et al., 1992; Palukaitis
and Garcia-Arenal, 2003). CP has been demonstrated to have roles in
encapsidation (Suzuki et al., 1991), systemic movement within infected plants
(Schmitz and Rao, 1998; Taliansky and Garcia-Arenal, 1995; Wong et al.,
1999), host range (Suzuki et al., 1991) and aphid transmission (Ng et al.,
2000; Perry et al., 1998).
Numerous strains of CMV have been classified into two major
subgroups (soubgroups I and II) on the basis of serological properties and
nucleotide sequence homology (Palukaitis et al., 1992). The subgroup has
been further divided into two groups (IA and IB) by phylogenetic analysis
(Rossinck et al., 1999; Palukaitis and Garcia-Arenal, 2003).
2.2.2 Cucumber leaf spot virus (possible Tombusvirus) (CLsV)
CLsV has been reported in Europe (Germany, Greece, and Great Britain) and
Jordan (Weber, 1986). Cucumber is the only natural host known for CLsV.
Irregularly shaped spots, at first pale -green to yellowish, which later have
necrotic centers occur in infected cucumber leaves. Infected plants grow
slowly. Experimentally infected hosts include 16 species in 5 dicot families
(Weber, 1982). Celosia argentea with red-brown necrotic spots without
systemic infection and Chenopodium quinoa with local necrotic spots without
systemic infection are suitable for assay and diagnosis (Weber, 1986).
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The CLsV isometric particles of 28-nm diameter have a sedimentation
coefficient of 127 S (Weber, 1982). The type strain of CLsV contains a single-
stranded RNA of 1650 kDa., according for 20% of the particle weight, and a
single polypeptide coat protein of 44 kDa. CLsV has strong immunogenicity.
Cucumber fruit streak virus is considered a separate strain of this virus
(Weber, 1986).
In Bulgaria, CLsV produces sporadically, chlorotic spots, sometimes
with necrotic centers on the leaves Aureus. Later, symptoms were less
recognizable or they disappeared completely. The 664-nt amplicon sequence
had 95% nucleotide and 98% amino acid sequence identity with the Spanish
CLSV isolate (Accession No. AY038365) and 98 and 99% identity,
respectively, with another CLSV isolate. The nucleic acid sequence of the
Polish CLSV isolate was 81-84% identical to the equivalent region of two
isolates of Pothos latent virus, another aureusvirus (Accession No AJ243370
and X87115) and had 86% identity with the amino acid sequence of both
isolates. This virus has been reported in Poland, Germany, Great Britain,
Jordan, Greece, Saudi Arabia, Spain and Bulgaria (Pospieszny and Cajza,
2004). Full-length clones of the genome of the aureus virus and Cucumber
leaf spot virus (CLSV) have been determined by Reade et al.(2003).
2.2.3. Cucumber necrosis virus [possible Necrovirus (CNV)]
CNV was discovered in Canada in glasshouse cucumbers, the only natural
host. CNV causes necrotic spots, pronounced leaf malformations and
dwarfing of plants. CNV isometric particles of 31nm diameter are 86 kDa and
have a sedimentation coefficient of 133S. Viral RNA accounts for 16% of the
particle weight (Dias and McKeen, 1972). Serological tests enable reliable
detection of CNV in the crude sap of infected plants.
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Experimental, mechanical inoculations were successful in many
species in Amaranthaceae, Chenopodiaceae, Compositae, Leguminosae, and
Solanaceae. Suitable assay plants include Cucumis sativus, Gomphrena
globosa. Chenopodium amaranticolor (local necrotic lesions) (Dias and
McKeen, 1972).
The soil-borne CNV is preserved in infected plant parts in the soil,
wherefrom it is transmitted by zoopores of Olpidium cucurbitacearum, a
chytrid fungus, which also transmits the related Tobacco necrosis virus. The
related fungus, O. brassicae, does not transmit CNV (Dias and McKeen,
1972). Uncontaminated soil glasshouses is the primary measure to protect
against CNV.
Tombusviruses are small, plus-sense, single-stranded RNA viruses of
plants. RNA-dependent RNA polymerases of two tombusviruses, Tomato
bushy stunt virus (TBSV) and Cucumber necrosis virus (CNV), have been
partially purified from infected Nicotiana benthamiana plants (Nagy and
Pogany, 2002).
2.2.4. Cucumber green mottle mosaic virus (CGnMtMV)
CGnMtMv, widespread in Asia (India), Japan, and Europe, causes severe
damage in indoor cucumber production. The quality of fruits is reduced, yield
losses may be 155 and, in winter production, even 30% (Raj et al., 2007b).
In addition to cucumber, melon (Cucumis melo) and watermelon
(Citrullus vulgaris) are natural hosts of CGnMtMV, but does not infect the
Cucurbita pepo. Initial symptoms of CGnMtMV on cucumber leaves include,
mild vein clearing and wrinkling which develop into pale to dark green
mosaic. Leaves are small and malformed, especially indoors. Some strains of
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CGnMtMV cause pale-yellow to white line, ring, or star-shaped spots (Raj et
al., 2007b).
CGnMtMV particles are straight rods, 300 x 10 nm, with a coat protein
of 17.2 kDa and a single-stranded RNA that accounts for 6% of the particle
weight (Hollings et al., 1975).
To identify this virus, the universal primers is prCG1 (+)(-). Specific
oligonuleotide primers corresponding to the heat labile regions of CGMtMV
could be employed for rapid assay of virus variability (Kim et al., 2003).
A new tobamo-like virus was isolated from a greenhouse-grown
cucumber that showed severe mosaic distortion on leaves and fruit, in the
southern part of Japan. The virus was tentatively designated Cucumber mottle
virus (CuMoV) and further characterized. The size and antigencity of the coat
protein (CP) and the complete sequence of the genome were compared with
those of the known cucurbit-infecting tobamoviruses, the W and SH strains of
Cucumber green mottle mosaic virus (CGMtMV), the C and Y strains of
Kyuri green mottle mosaic virus (KGMtMV), Cucumber fruit mottle mosaic
virus (CFMtMV), and Zucchini green mottle mosaic virus (ZGMMV). The CP
of CuMoV migrated more slowly than those of CGMtMV-W and -SH and
KGMMV-C and -Y in sodium dodeyl sulfate polyacrylamide gel
electrophoresis. In Western blot analysis, the CP of CuMoV cross-reacted
weakly with antisera against CGMtMV-W and did not react with antisera
against KGMMV-Y. The overall nucleotide sequence of CuMoV had 62.5 to
63.5% identity with those of CGMtMV-W, -SH, KGMMV-Y, CFMMV, and
ZGMMV. The genome organization was characteristic of tobamoviruses,
encoding a 131-kb protein, a 188-kb protein, a movement protein (MP), and
CP in 5′ to 3′ order. In the phylogenetic analysis of the CP, CuMoV was
placed in a separate lineage from CGMtMV-W, -SH, KGMMV-C, -Y,
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CFMMV, and ZGMMV. The results indicate that CuMoV is a distinct
tobamovirus species which represents a third sub-subgroup in the cucurbit-
infecting tobamoviruses (Orita et al., 2007).
2.2.5. Melon necrotic spot virus (MnNcSpoV)
MnNcSpoV is common in melon and cucumber crops worldwide and can
substantially reduce yields (Bos et al., 1984). Melon and cucumber as natural
hosts of MnNcSpoV, express symptoms of disease as necrotic lesions and
spots on leaves and stem necrosis on melon. Experimentally infected
cucurbitaceous hosts that react differently to isolates of varying origin include
Citrullus lanatus, Cucurbita moschata, Lagenaria siceraria, and others.
Diagnostic plants include Cucumis melo with necrotic lesions in inoculated
cotyledons followed by systemic infection, and Citrullus lunatus with dark
local lesions on inoculated leaves, without systemic infection (Hibi and
Furuki, 1985).
The MnNcSpoV isometric particles consists of 30 nm diameter and
have a sedimentation coefficient of 134S. The coat protein consists of a single
46-kDa polypeptide, and the single-stranded RNA accounts for about 17.8%
of the particle weight (Hibi and Furuki, 1985). MnNcSpoV is
immunogenically active. Insufficient comparative data are available to
designate strains of some isolates which differ slightly based on
symptomatology and host range (Hibi and Furuki, 1985).
New strains of Melon necrotic spot virus (MNSV), designated MNSV-
YS and MNSV-KS, caused much more severe growth retardation on melon
plants than MNSV-NH, which was previously reported as the most severe
strain of MNSV in Japan. The complete sequences of MNSV-YS and MNSV-
KS were determined, and an RT-PCR-RFLP method based on these sequences
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was successfully developed to detect and discriminate between the three
strains (Kubo et al., 2005).
2.2.6. Watermelon mosaic potyvirus 2 (WmMV2)
WmMV2 is distributed worldwide and ranks as a serious problem in cucurbit
production. WmWV2 causes economic damage in watermelon and other
susceptible cucurbits, and is reflected mostly as reduced plant growth, yield
losses, and a decrease in fruit quality (Raj et al., 2007b).
Most natural hosts of WmMV2 are in the gourd family. In addition to
watermelon (Citrullus lanatus), melon (C. melo), courggette (Cucurbita pepo)
pumpkin (C. pepo) and squash (C. maxima). Beans and peas are important
natural hosts among legumes. WmMV2 in nature infects different leguminous,
malvaceous and chenopodiceous weeds, crops, and ornamentals (Purcifull et
al., 1984). More than 160 dicot species in 23 families are experimental virus
hosts (Molnar and Schmelzer, 1964). Diagnostic plants include Chenopodium
amaranticolor with chlorotic and necrotic local lesions and Lavatera
trimestris with necrotic local lesions.
The WmMV2 filamentous particles of 760 nm consist of a coat protein
subunit of 34 KDa and RNA that sediments at 39S (Purcifull et al., 1984).
WmMV2 has good antigenicity. WmMV2 was described separately to
distinguish it from the earlier described watermelon mosaic virus 1, which is
now designated as a strain of papaya ringspot virus (Purcifull and Hiebert,
1979).
The initial symptom of WmMV2 is mild chlorosis at watermelon
leaves, followed by mosaic and leaf distortion. Green mosaic along the veins
and/or green bubble-like protuberances on chlorotic interveinal leaf parts
occur as disease develops. WmMV2 infection early in the season causes poor
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development of young leaves, internode shortening, show plant growth, and
reduced fructification.
2.2.7. Squash mosaic comovirus (SqMV)
SqMV occurs naturally in cucurbits, primarily in squash, cucumber, courgette,
pumpkin, melon and watermelon. Infected plants may remain symptomless or
show different symptoms, such as mosaic, blister mottle, ring patterns,
enations, and leaf deformations, and/in the case of early, severe infections the
fruits are also malformed (Campbell, 1971). Experimental infections have
been recorded in hosts in five families (Freitag, 1956).
SqMV consists of three isometric particles (T.M. and B) of 30-nm
diameter with sedimentation coefficients of 57S(T), 95S(M), and 118S(B) and
molecular weights of 4500 (T), 6100 (M), and 6900(B) kDa (M) 2400 kDa
(B), accounting for about 26.8% (M) and 34.8% (B) of the particle weight.
The B component is infective, but other components require further
investigation (Raj et al., 2007b).
The TIP of SqMV is 70 to 800C the DEP is 1 x 10-4 to 1 x 10-6 and LIV
at 200C is 4 weeks (Campbell, 1971). Strains are designated based on
differences in host reaction, that is, muskmelon mosaic virus and cucurbit
mosaic virus (Stace-Smith, 1981). Various isolates of SqMV belong to two
serologically distant strains; strain I (but not strain 2) isolates experimentally
infect cantaloupe melon (Nelson and Knuhtsen, 1973). SqMV is strongly
immunogenic (Stace-Smith, 1984).
The uncontrolled introduction of infected seed has contributed greatly
to the wide distribution of SqMV in several countries. SqMV causes
economical damage resulting from severe systemic infections (Raj et al.,
2007b).
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2.2.8. Zucchini yellow mosaic potyvirus (ZYMV)
Cucurbits are natural hosts of ZYMV, including cucumber, courgette, melon,
watermelon, and zucchini squash. Symptoms in infected plants vary,
depending on virus strain and plant cv. (Lecoq, 1981; Lesemann et al., 1983).
Symptom types in infected leaves include vein clearing. Yellowing and
enations, infected fruits are malformed, with mixed pulp and distorted seeds,
and severe systemic infections cause plant dwarfing. Plants in 11 families have
been experimentally infected (Lisa and Lecoq, 1984). Diagnostic plants
include Chenopodium amaranticolor and C. quinoa with chlorotic local
lesions, but no systemic infection, and Gomphrena globosa with local lesions,
but no systemic infection.
The ZYMV filamentous particles of 750 nm consist of a 36-kDa coat
protein and 2930-kDa single stranded RNA (Lisa, 1981). Several strains of
ZyMV have been described based on symptoms in individual hosts. ZYMV
has good immunogenicity (Lisa and Lecoq, 1984). ZYMV, first described in
the Mediterranean, is considered to be spread worldwide. Zhao et al. (2003)
alos reported the presence of ZYMV in the China and he grouped this virus on
the basis of phylogenetic analysis.
A reverse transcription-polymerase chain reaction (RT-PCR) method
was used to identify Zucchini yellow mosaic virus (ZYMV) in leaves of
infected cucurbits. Oligonucleotide primers which annealed to regions in the
nuclear inclusion body and the coat protein (CP) genes, generated a 300-bp
product from ZYMV and also from the closely related Watermelon mosaic
virus type 2 (WMV-2) 1186-bp amplified product was obtained for ZYMV. .
Nucleotide sequence analysis of the 300-bp fragments of Australian ZYMV
and WMV-2 strains revealed 93.7–100% sequence identity between ZYMV
strains. Multiple sequence alignments indicated that the nucleotide sequence
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which codes for the N-terminus of the CP was 74–100% identical for different
isolates of ZYMV. The Australian isolate of WMV-2 was 43–46% identical to
all isolates of ZYMV and was 84.6% identical to a Florida isolate of WMV-2
(Thomson et al., 1995).
2.2.9. Zucchini yellow fleck virus (ZYfkV)
ZYFkV is less common in Spain and Greece (Martelli, 1988). In nature, it
infects courgette plants, first causing various yellow spots on the leaves that
later coalesce to produce a general yellowing and necrosis. Plants severely
infected with ZYFkV early in the season grow slowly and yields are reduced
substantially. Other natural hosts include cucumber, melon, and watermelon,
and experimentally infected hosts belong to the Cucurbitaceae family.
Marrow with necrotic lesions followed by systemic yellow flecks and
Lagenaria siceraria with systemic vein clearing are suitable test for assay
(Martelli, 1988).
Molecular evidence proved that Zucchini yellow fleck virus is a distinct
and variable potyvirus related to Papaya ring spot virus and Moroccan
watermelon mosaic virus (Desbiez et al., 2007).
Aphid vectors transmit ZYFkV non-persistently. ZYFkV can also be
transmitted by contact with infective plant sap.
2.2.10. Geminivirus
The family Geminiviridae is one of the largest groups of plant viruses. The
morphology of Geminivirus particles is unique and they are characterized by
geminate shape and the small size ~ 30 x 20 nm. They have a circular single
stranded DNA genome which replicates in the host cell nucleus. The
transmission of these viruses by the insect vectors is in a persistent manner.
They have the propensity to infect phloem cells (Sunter et al., 1994; Harrison
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and Robinson, 1999; Varma and Malathi, 2003). Geminiviruses infect a wide
range of weeds and cultivated plants, including both monocots and dicots. The
infections can affect plants in many ways. One of the physiological processes
seriously affected is photosynthesis with decreasing yields of starch as a result.
Geminiviruses also disrupt flower and fruit formation in crops such as tomato,
pepper and cotton (Moffat, 1999).
The genome organization and biological properties of Geminiviruses
allow them to be divided into four genera. Those that have a monopartite
genome and are transmitted by leafhoppers in monocotyledonous and
dicotyledonous plants are members of the genus Mastrevirus, of which Maize
streak virus (MSV) is the type species. The genus Curtovirus comprises
viruses that have a monopartite genome and are transmitted by leafhoppers in
dicotyledonous plants; Beet curly top virus (BCTV) is the type species. The
genus Topocuvirus has only one member (the type species), Tomato pseudo-
curly top virus (ToPCTV) which has a monopartite genome and is transmitted
by treehoppers in dicotyledonous plants. The fourth genus, Begomovirus,
includes viruses that are transmitted by whiteflies to dicotyledonous plants;
Bean golden mosaic virus (BGMV) is the type species.
These viruses are responsible for a significant amount of crop damage
worldwide. Epidemics of Geminivirus diseases have arisen due to a number of
factors, including the recombination of different Geminiviruses co-infecting a
plant, which enables novel, possibly virulent viruses to be developed. Other
contributing factors include the transport of infected plant material to new
locations, expansion of agriculture into new growing areas, and the expansion
and migration of vectors that can spread the virus from one plant to another.
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2.2.11. Begomovirus (Bean golden mosaic virus)
Begomovirus have emerged as constraints to the cultivation of a variety of
crops in various parts of the world. Diseases caused by Begomovirus poses a
serious threat to sustainable agriculture, particularly in the tropics and sub-
tropics. Another concern is the emergence of diseases that are caused by a
complex of Begomovirus and satellite DNA molecules (Saunders et al., 2001;
Varma and Malathi, 2003; Bull et al., 2004; Stanley, 2004).
Some crops appear to be a paradise for Begomovirus. So far, 45
recognized and 30 tentative species of Begomovirus have been found to
naturally infect tomato, pepper and cucurbits in the New and Old world. Some
of the viruses have a large number of distinct strains (Jones, 2003).
Begomovirus have bipartite genomes (A and B components), with
some exceptions [e.g., Tomato yellow leaf curl virus (TYLCV), Cotton leaf
curl virus (CLCuV), Tomato leaf curl virus (ToLCV)] for which no B
components has been found (Fauquet et al., 2003).
Begomovirus (type species Bean golden mosaic virus) is the only
genus of the family to be either bipartite with virus genes resident on two
different circular ssDNA molecules (DNA A, DNA B) each of about 2.6-2.8
kb, or monopartite with all genes resident on one (DNA A-like) ss DNA of
about 2.8 kb. The twinned particles have diameter of 18-20nm, 30 nm long,
like most of the Geminiviruses. The Begomovirus are all transmitted by the
whitefly B. tabaci in a circulative manner an d infecting dicotyledonous
plants. They have been considered as the most numerous and widespread
group of whitefly-transmitted viruses causing severe epidemics in India. The
fast spreading Begomovirus in India on cucurbits is Tomato leaf curl
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NewDelhi virus (Soharab et al., 2003, 2006) and Squash leaf curl China virus
(Singh et al., 2008).
2.2.11.1. Tomato leaf curl virus
Tomato yellow leaf curl virus (TYLCV) is one of the most devastating viruses
of cultivated tomatoes in tropical and subtropical regions. Depending on the
timing of infection, yield losses can reach up to100%. In many tomato-
growing areas, TYLCV has become the limiting factor for production of
tomatoes in both open field and protected cultivation systems (Lapidot and
Friedmann, 2002). Although TYLCV is primarily known as a pathogen of
tomato, epidemics have been reported in other crops (Navas-Castillo et al.,
1999). TYLCV can have a catastrophic impact on tomato production and is
often associated with severe crop losses both in the eastern Mediterranean and
the Western Hemisphere (Al-Musa, 1982; Cohen and Nitzany, 1966; Polston
and Anderson, 1997).
TYLCV belongs in the family Geminiviridae, genus Begomovirus.
TYLCV is a monopartite Begomovirus. Two strains of TYLCV have been
reported in Israel: TYLCV (Acc. No. X15656) and TYLCV-Mld (Acc. No.
X76319) (Antignus and Cohen, 1994; Navot et al., 1991). TYLCV-Mld
produces symptoms in susceptible tomato cultivars that are indistinguishable
from those produced by TYLCV. TYLCV is reported to be a recombinant
virus between TYLCV-Mld and an ancestor of Tomato leaf curl virus
(ToLCV), a Begomovirus described from India (Harrison and Robinson, 1999;
Navas-Castillo et al., 2000). TYLCV possesses a portion, while the rest of the
genome is nearly identical to that of TYLCV-Mld.
The Rep (Replication associated protein) gene is essential for viral
DNA replication and is involved in transcriptional regulation (Fontes et al.,
1994; Lazarowitz, 1992). The TrAP (Transactivation protein) gene has been
2: Review of Literature
26
shown to play a role in systemic infection and the suppression of host defense
responses (Etessami et al., 1991; Bisaro et al., 1999; Brough et al., 1992). The
REn (Replication enhancer protein) gene enhances replication (Sunter et al.,
1990; Etessami et al., 1991). C4 appears to play a role in pathogenicity (Krake
et al., 1998) and V2 has been shown to play a role in virus movement (Wartig
et al., 1997). The TYLCV coat protein gene is the most abundant protein
produced by TYLCV (Timmermans et al., 1994). This protein is required for
whitefly transmission, binds to viral single stranded DNA (ssDNA), contains a
nuclear targeting signal which mediates movement of viral nucleic acid into
the host cell nucleus, and may play a role in systemic movement (Azzam et
al., 1994; Briddon et al., 1990; Kunik et al., 1998; Palanichelvam et al., 1998;
Palmer and Rybicki, 1998). The replication strategy used by TYLCV consists
of the conversion of ssDNA into a double-stranded DNA (dsDNA)
intermediate, followed by the use of dsDNA as a template to produce ssDNA
genomes by a rolling-circle replication mechanism (Gutierrez, 1999; Hanley-
Bowdoin et al., 1999).
Until about 1990, TYLCV was recognized as a pathogen in the eastern
Mediterranean, and was found in tomato fields in Cyprus, Egypt, Jordan,
Israel, Lebanon, Syria, and Turkey (Czosnek and Latterrot, 1997) although it
was first described in Israel (Cohen and Nitzany, 1966; Cohen, and Antignus,
1994). However, over the last decade, the geographic Mediterranean, the
Caribbean, Mexico and the southeastern U.S. (Moriones and Navas-Castillo,
2000). TYLCV appeared in the eastern Caribbean in the late 1980’s, and was
found for the first time in tomato in Cuba in 1989, the Dominican Republic in
1992, Jamaica in 1993, The Bahamas in 1996, and Puerto Rico in 2001 (Bird
et al., 2001; McGlashan et al., 1994; Polston et al., 1994; Ramos et al., 1996;
Sinisterra et al., 2000; Nakhla et al., 1994; Martinez-Zubiaur et al., 1996). In
the western Caribbean it was found in Yucatan, Mexico in 1997 (Ascecio-
2: Review of Literature
27
Ibanez et al., 1999). TYLCV was found for the first time in the United States
in Florida in 1997, in Georgia in 1998, Louisiana in 2000, Mississippi and
North Carolina in 2001, and Alabama and South Carolina in 2005 (Ingram and
Henn, 2001; Ling et al., 2006; Momol et al., 1999; Pappu et al., 2000; Polston
et al., 1999; Valverde et al., 2001).
Although, there are no estimates of the economic impact of TYLCV,
this virus is responsible for crop failures in tomato in many locations around
the world, and in fact TYLCV has become the limiting factor in tomato
production in many growing areas.
Amplification with the polymerase chain reaction (PCR) is the most
sensitive assay for the detection and amplification of nucleic acids today. The
rapid improvement and availability of different DNA polymerases for use in
PCR, combined with a small closed circular ssDNA and a published sequence,
make it relatively easy to design overlapping or nearly overlapping primers to
amplify the full TYLCV genome in a single reaction. Two nearly overlapping
primers, which bind in the intergenic region (IR) of the TYLCV genome, were
used to amplify a genomic size fragment of TYLCV from total DNA extracted
from an infected plant. By using different primers complementary to different
regions of the viral genome, they have demonstrated the amplification of
subgenomic fragments of the viral genome from total DNA extracted from
either infected plants or from viruliferous whiteflies (Navot et al., 1992). A
large number of TYLCV specific primers were designed for the PCR
amplification of different parts of the viral genome from infected plants as
well as from whiteflies (Atzmon et al., 1998; Mehta et al., 1994; Navas-
Castillo et al., 1998; Lapidot and Friedmann, 2002)
It is now understood that the tomato yellow leaf curl disease (TYLCD)
is induced by a large number of related viruses (Moriones and Navas-Castillo,
2: Review of Literature
28
2000). Moreover, a number of viruses inducing TYLCD can co-exist in the
same geographical region. Thus, a detection assay that discriminates among
the different viruses, preferably in a single reaction, is highly desirable. One
approach was to develop two sets of PCR primers, one designed to amplify
DNA from a wide array of TYLCD-causing viruses, and another that
specifically amplifies the DNA of Tomato yellow leaf curl Sardinia virus
(TYLCSV) (Martinez-Culebras et al., 2001). In addition, both primer sets
were able to be used simultaneously in a single duplex PCR amplification
reaction, producing both amplification products for TYLCV and for TYLCSV
(Martinez-Culebras et al., 2001). In a similar approach, three sets of PCR
primers were designed which amplified individually and specifically TYLCV,
TYLCSV, and TYLCV-Mld (Anfoka et al., 2005). The PCR primers were
designed in such a way that each set produced a different size product
following amplification of the respective target virus. The triplex PCR
amplification was shown to amplify one or all three viral products in a single
reaction, and due to the different sizes of each amplified product, it was
possible to discriminate among the three viruses in a single reaction (Anfoka
et al., 2005).
In India, four species of ToLCVs, two form northern and southern
India, have been reported to cause ToLCNDV in various parts of India. They
are Tomato leaf curl New Delhi virus (ToLCNDV) (Srivastava et al., 1995;
Padidam et al., 1995), Tomato leaf curl Gujarat virus (ToLCGV)
(Chakraborty et al., 2003), Tomato leaf curl Bangalore virus (ToLCBV)
(Hong and Harision, 1995; Muniyappa et al., 2000; Kirthi et al., 2002) and
Tomato leaf curl Karnataka virus (Chatchawankanphanich and Maxwell,
2002; Fauquet et al., 2008).
2: Review of Literature
29
Table 2.2. Tomato leaf curl virus- Indian and Pakistan isolates.
Virus Host Place Accession no.
Strain
Tomato leaf curl virus - Bangalore 1
- Bangalore
Z48182 ToLCBV-A
Tomato leaf curl Bangalore virus - A
Tomato Kerala DQ887537 ToLCBV-A
Tomato leaf curl Bangalore virus - A
Tomato Kolar AF428255 ToLCBV-A
Tomato leaf curl Bangalore virus - B
- - AF295401 ToLCBV-B
Tomato leaf curl Bangalore virus - B
Cotton Fatehabad AY456684 ToLCBV-B
Tomato leaf curl Bangalore virus – C
- Bangalore AF165098 ToLCBV-C
Tomato leaf curl Bangalore virus - C
- Bangalore AY428770 ToLCBV-C
Tomato leaf curl Gujarat virus
- Mirzapur AF449999 ToLCGV--[IN:Mir:99]
Tomato leaf curl Gujarat virus
Tomato Vadodara AF413671 ToLCGV-[IN:Vad:99]
Tomato leaf curl Gujarat virus
Tomato Varanasi AY190290 AY190291
ToLCGV-[IN:Var:01]
Tomato leaf curl Gujarat virus
Tomato Nepal AY234383 ToLCGV-[NP:Pan:00]
Tomato leaf curl Joydebpur virus
Chili Kalyani EF194765 ToLCJoV-IN
Tomato leaf curl Kerala virus
Tomato Kerala DQ852623 ToLCKeV-[IN:KerII:05]
Tomato leaf curl Karnataka virus
Tomato Bangalaore U38239 ToLCKV-Ban[IN:Ban:93]
Tomato leaf curl Karnataka virus
Tomato Janti AY754812 ToLCKV-Jan[IN:Jan:05]
Tomato leaf curl New Delhi virus
Tomato Bangladesh & Jessore
AJ875157 AJ855158
ToLCNDV-IN[BG:Jes:Svr :05]
Tomato leaf curl New Potato Merrut EF043231 ToLCNDV-
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30
Delhi virus EF043232 IN[IN:Hap:Pot:05] Tomato leaf curl New Delhi virus
Potato Happur EF043230 EF043233
ToLCNDV-IN[IN:Hap:Pot:05]
Tomato leaf curl New Delhi virus
Cotton Hissar EF063145 ToLCNDV-IN[IN:His:Cot:05]
Tomato leaf curl New Delhi virus
Tomato Lucknow X89653 ToLCNDV-IN[IN:Luc]
Tomato leaf curl New Delhi virus
Potato Meerut AY286316 AY158080
ToLCNDV IN[ IN:Mee:Po12:02]
Tomato leaf curl New Delhi virus
Tomato New Delhi DQ169056 DQ169057
ToLCNDV-IN[IN:ND:05]
Tomato leaf curl New Delhi virus
- New Delhi AY428769 AY438563
ToLCNDV-IN[IN:ND:AVT1]
Tomato leaf curl New Delhi virus
- New Delhi U15016 ToLCNDV-IN[IN:ND:Mld:92]
Tomato leaf curl New Delhi virus
- New Delhi U15015 U15017
ToLCNDV-IN[IN:ND:Svr:92]
Tomato leaf curl New Delhi virus
Luffa Sonepat AY939926 AY939924
ToLCNDVIN[ PK:Dar:T5/6:01]
Tomato leaf curl New Delhi virus
- Pakistan AF448058 AY150305
ToLCNDVIN[ PK:Dar:T5/6:01]
Tomato leaf curl New Delhi virus
- Pakistan AF448059 AY150304
ToLCNDV-IN[PK:Isl:T1/8:00]
Tomato leaf curl New Delhi virus
Chili Pakistan DQ116880 DQ116882
ToLCNDV-IN[PK:Kha:Chi:04]
Tomato leaf curl New Delhi virus
Luffa Pakistan AM292302 ToLCNDV-IN[PK:Mul:Luf:04]
Tomato leaf curl New Delhi virus
Solanum nigrum
Pakistan AJ620187 AJ620188
ToLCNDV-IN[PK:Sn:97]
Tomato leaf curl New Delhi virus
Solanum nigrum
Pakistan DQ116885 ToLCNDV-IN[PK:Sol:04]
Tomato leaf curl New Delhi virus
Solanum nigrum
Pakistan DQ116883 ToLCNDVIN[ PK:Sol:PT10:04]
Tomato leaf curl New Delhi virus
- Pakistan AM258977 AM392426
ToLCNDV-IN[PK:Lah:04]
Tomato leaf curl New Delhi virus- Papaya
Papaya New Delhi DQ989325 ToLCNDV-Pap[IN:ND:Pap:05]
2: Review of Literature
31
Tomato leaf curl Pune virus
- Pune AY754814 ToLCBV-[IN:Pun:05]
Tomato leaf curl Rajasthan virus
- Rajasthan DQ339117 ToLCBV-[IN:Raj:05]
- = Host not known (Source: NCBI search)
Table 2.3. Natural host plants of Tomato leaf curl New Delhi virus
(ToLCNDV) and their distribution.
S.N. Host species Country Reference/Accession No. 1. Tomato India Srivastava et al., 1995, Padidam et al.,
1995 Tomato Pakistan Mansoor et al., 1997; Shih et al.,
2003, Hussain et al., 2005 Tomato Bangladesh Maruthi et al., 2005 2. Chilli India Khan et al., 2006; Reddy et al., 2005 Chilli Pakistan Hussain et al., 2004 3. Luffa India Sohrab et al., 2003 Luffa Thailand AF102276 4. Watermelon Pakistan Mansoor et al., 2000 5. Potato India Usharani et al., 2004 6. Bitter gourd Pakistan Tahir and Haider, 2005 7. Bottle gourd India DQ272539/DQ272540 8. Pigeon pea India Raj et al., 2005a 9. Cowpea India Reddy et al., 2005 10. Croton India Reddy et al., 2005 11. Eclipta Pakistan Haider et al., 2006
2: Review of Literature
32
2.2.12. Cucurbit yellow stunting disorder virus (CYSDV)
The high populations of B. tabaci and its preference for cucurbits and the
possible displacement of T. vaporariorum in greenhouses, can account for the
higher incidence of CYSDV and its predominance over BPYV. In countries of
the Mediterranean basin such as Spain, Turkey and Portugal, CYSDV is the
main crinivirus present and is considered to be the major causal agent of
cucurbit yellowing disease (Ce´lix et al., 1996; Berdiales et al., 1999; Rubio et
al., 1999). In Spain, approximately 75% of the cucumber samples tested were
found to be infected by CYSDV and only 2% by BPYV. This suggests that
BPYV, although present in Spanish greenhouses since 1980s, has been
practically displaced by CYSDV (Ce´lix et al., 1996). CYSDV was also the
major cause of a yellowing disorder in glasshouse cucumbers in Lebanon
(Abou-Jawdanh et al., 2000). Greece is the only Mediterranean country with a
different situation, where data concerning the criniviruses observed indicate
that T. vaporariorum are probably more abundant in greenhouse crops during
winter and spring (Dovas et al., 2002) and BPYV is the predominant crinivirus
in melon and cucurbit crops with CYSDV being present only in Rhodes and
Crete islands, and in the Peloponnese area in southern Greece (Katis et al.,
2001). CYSDV, BPYV and CABYV reported in Cyprus for the first time.
CVYV, a tentative species of the genus Ipomovirus (Lecoq et al., 2000) also
transmitted by B. tabaci (Harpaz and Cohen, 1965; Mansour and Al-Musa,
1993), was detected in greenhouse-grown cucumber crops. CVYV affects
cucumbers and other cucurbit crops in the Mediterranean Basin (Harpaz and
Cohen, 1965; Al-Musa et al., 1985; Yilmaz et al., 1989; Cuadrado et al.,
2001) but its incidence is low. Its incidence in Cyprus is also low, possibly
indicating poor transmission efficiency and/or persistence of the virus within
the vector. ZYMV was also detected in few greenhouse cucumber crops and
therefore is of limited.
2: Review of Literature
33
High mobility of whiteflies feeding on many plant species and their
high multiplication rate are probably the reasons for the rapid spread of
whitefly-transmitted closteroviruses once they are introduced in a given area.
The majority of whitefly-transmitted closteroviruses have a clear vector
specificity generally confined to a single whitefly species (Karasev, 2000).
The group of whitefly-transmitted closteroviruses inludes: Cucurbit yellow
stunting disorder virus (CYSDV), Abutilon yellows virus (AbYV), Beet
pseudo-yellows virus (BPYV), Cucumber yellows virus (CuYV), Lettuce
chlorosis virus (LCV), Lettuce infectious yellows virus (LIYV), Potato yellow
vein virus (PYVV), Strawberry pallidosis associated virus (SPaV), Sweet
potato chlorotic stunt virus (SPCSV), Tomato chlorosis virus (ToCV) and
Tomato infectious chlorosis virus (TICV), all belonging to the genus
Crinivirus. Tentative species in this genus is Diodea vein chlorosis virus
(DVCV) (Martelli et al., 2002; Livieratos et al., 2004; Tzanetakis and Martin,
2004; Tzanetakis et al., 2005). CuYV was postulated to be a strain of BPYV
(Wisler et al., 1998).
CYSDV, as a member of the genus Crinivirus (Martelli et al., 2002),
consists of viral particles that are flexible rods with lengths between 750 to
800 nm (Liu et al., 2000). CYSDV has a narrow host range limited to species
of the family Cucurbitaceae in which it is confined to phloem-associated cells
(Célix et al., 1996; Marco et al., 2003). Under experimental conditions
CYSDV cannot be inoculated mechanically, therefore controlled-inoculation
has to be carried out using whiteflies or by graft-inoculation (López-Sesé and
Gómez-Guillamón, 2000; Marco et al., 2003). The CYSDV genome consists
of two molecules of ssRNA of positive polarity designated RNA1 and RNA2
(Célix et al., 1996).
CYSDV, as a whitefly-transmitted virus, affects extensively cucurbit
crops in many warm and temperate areas of production (Hassan and Duffus,
2: Review of Literature
34
1991; Wisler et al., 1998). It is able to infect members of the family
Cucurbitaceae, like melon, cucumber, marrow, squash and watermelon. It was
first described as a disease of cucurbit crops in the United Arab Emirates in
1982 (Hassan and Duffus, 1991). In 1996, it was reported to appear on melon
and cucumber plants in Spain (Célix et al., 1996), in 1999 on melon in North
America (Kao et al., 2000), Portugal (Louro et al., 2000) and Morocco
(Desbiez et al., 2000), in 2000 on cucumber and melon plants in Lebanon
(Abou-Jawdah et al., 2000) and in 2001 on melon in Southern France (Desbiez
et al., 2003). Using probes made to a portion of CYSDV HSP70 and CP
genes, CYSDV was identified in cucurbits from Jordan, Turkey, Egypt, Spain
and Israel (Rubio et al., 1999, 2001).
The phloem confinement of CYSDV and its low titer in infected plants
made this virus difficult to isolate and consequently, antisera against virions
have not been produced. However, an antiserum against recombinant CYSDV
coat protein expressed in Escherichia coli has been produced by Hourani and
Abou-Jawdah, (2003). This antiserum could be successfully used in tissue-blot
and dot-blot inmunoassays, and also in indirect and DAS-ELISA (Hourani and
Abou-Jawdah, 2003). On the other hand, using a digoxygenin (DIG)-labelled
RNA probe, produced by in vitro transcription, CYSDV RNA can be detected
by hybridization either in tissue prints of plant stems, petioles and major leaf
veins or by dot blot of total RNA extracts from leaf samples (Marco et al.,
2003). Moreover, (DIG)-labelled cDNA probes allowed CYSDV detection
and quantification in B. tabaci extracts (Ruiz et al., 2002). Additionally, using
primers complementary to different portions of the CYSDV genome, it is
possible to detect CYSDV by RT-PCR analysis in its different hosts (Marco et
al., 2003; Rubio et al., 1999, 2001).
2: Review of Literature
35
2.2.13. Cucurbit aphid-borne yellows virus
During the 80´s, yellowings were associated with the whitefly Trialeurodes
vaporariorum vectoring the crinivirus Beet pseudo-yellows virus (BPYV)
(Jordá et al., 1993). During the 90´s, T. vaporariorum was progressively
displaced by Bemisia tabaci and consequently, Cucurbit yellow stunting
disorder virus (CYSDV), a new crinivirus, spread over cucurbit crops (Célix
et al., 1996). Recently, Cucurbit aphid-borne yellows virus (CABYV) have
been detected in protected and open field melon (Cucumis melo), cucumber
(Cucumis sativus), squash (Cucurbita pepo) and watermelon (Citrullus
lanatus) crops of southeastern Spain, with a very high incidence (Juárez et al.,
2004).
CABYV is a member of the genus Polerovirus of the family
Luteoviridae (Mayo and D‘Arcy, 1999). Its viral particles are isometric,
approximately 25 nm in diameter, and encapsidate the CABYV genome,
which consists of a single stranded (ss) positive sense RNA molecule of 5.7 kb
which has neither a 5´-cap nor 3´poly(A) tail. The CABYV genome has been
fully sequenced (Guilley et al., 1994) and a full-length infectious clone is
available (Prüfer et al., 1995). It contains six major open reading frames
(ORFs), which are separated by an internal non-coding region (ca. 200
nucleotides) into a relatively divergent group of 5´-proximal genes and a more
conserved 3´-proximal gene block. The three 5´-proximal ORFs are expressed
from the genomic RNA and yield P1, P2 and the translational frame shift
protein P2-3. The three 3’-proximal ORFs (ORFs 4-6) are translated from a
sub genomic RNA and yield the viral capsid proteins (CP and RT) and P5. A
range of different translational strategies is used by CABYV to express these
proteins, including bypass of the first AUG codon and initiation at the second
AUG codon (P2 and P5), translational frame shift (-1) in the overlap region
2: Review of Literature
36
between ORF2 and ORF3 (P2-3) and read-through suppression of the UAG
termination codon of ORF4 (RT). P1 has recently been shown to be a
suppressor of gene silencing (Pfeffer et al., 2002). P2 and P2-3 contain
chymotrypsin-like protease and RNA-dependent RNA polymerase (RdRp)
domains, respectively, and are putative components of the RdRp complex
(Guilley et al., 1994). The ORF4 gene product is the major component of the
viral capsid, and the read-through (RT) protein is a minor structural protein
possibly involved in virus-vector interactions. While the role of CABYV P5
remains unclear, the corresponding homologous protein of Potato leaf roll
virus (PLRV), the type species of the genus Polerovirus, has been described as
the movement protein (Taliansky et al., 2003). Among poleroviruses, CABYV
has closest homology with Beet western yellows virus (BWYV), particularly
in ORFs 3 and 4 (Guilley et al., 1994).
CABYV is transmitted in a persistent and circulative manner by two
aphid species, the black melon aphid Aphis gossypii and the green peach aphid
Myzus persicae (Lecoq et al., 1992). The route of CABYV virions through
their vectors has been analyzed at an ultra structural level (Reinbold et al.,
2003). CABYV cannot be mechanically transmitted, and no other transmission
method has been described for this virus. The host range of CABYV includes
the major cultivated cucurbit species (melon, cucumber, squash and
watermelon), in which it remains confined to their phloem. However, there are
other non-cucurbit species of high agronomic importance which act as hosts
for CABYV, such are lettuce (Lactuca sativa) and fodder beet (Beta vulgaris)
(Lecoq et al., 1992). In addition, a number of weeds, which may serve as
reservoirs and primary sources of infection, can host CABYV. These include
Ecballium elaterium, Bryonia dioica, Senecio vulgaris, Capsella bursa-
pastoris, Crambe abyssinica, Papaver rhoeas, Montia perfoliata and Lamium
amplexicaule (Lecoq, 1999).
2: Review of Literature
37
CABYV was first described by H. Lecoq and co-workers in 1992 in
France where it affected open field cucurbit crops (Lecoq et al., 1992).
Afterwards, CABYV was detected in Italy, Greece, Tunisia, Algeria, Libano,
Turkey, Sudan, Nepal, Taiwan, China, Reunion Island, Swaziland, Brazil,
Honduras, Spain, California (U.S.A) and Cyprus (Abou-Jawdah et al., 1997;
Juárez et al., 2004; Lecoq, 1999; Lecoq et al., 1992, 2003; Lemaire et al.,
1993; Mnari-Hattab et al., 2005; Papayiannis et al., 2005). Extensive surveys
conducted by H. Lecoq and co-workers have shown that CABYV is one of the
most common cucurbit viruses in open field crops of a great diversity of areas
and environments (Lecoq, 1999; Lecoq et al., 2003).
CABYV can be readily diagnosed by ELISA. Excellent antisera have
been produced by Lecoq et al. (1992) to our knowledge, CABYV antisera
have not been produced on a commercial basis.
For RT-PCR, the mostly used oligonucleotides are 5´-
GAATACGGTCGCGGCTAGAAATC-3´ (CE9) and 5´-
CTATTTCGGGTTCTGGACCTGGC-3´ (CE10), identical and
complementary, respectively. For detection of CABYV in tissue prints, plant
stems, petioles and major leaf veins were cut with a razor blade and,
immediately after cutting, the cross-sections were blotted onto positively
charged nylon membranes (Roche Diagnostics). For detection of CABYV in
dot-blots, 0.5 µl of total RNA extracts prepared as in Célix et al. (1996) was
spotted onto positively charged nylon membranes. Both, tissue prints and dot-
blots were subsequently submitted to the same protocols. Briefly,
prehybridization and hybridization of membranes were carried out at 68ºC in
standard buffer (50% formamide, 5x SSC, 0.1% N-Laurylsarcosine, 0.02%
SDS, 2% Blocking Reagent.
2: Review of Literature
38
2.2.14. Mastrevirus (Maize streak virus)
The type member is Maize streak virus (MSV). It includes leafhopper-
transmitted viruses having single-component genomes (monopartite) infecting
monocotyledonous species in most cases (Wheat dwarf virus, WDV; Digitaria
streak virus, DSV) with few exceptions namely Tobacco yellow dwarf virus
(TYDV) and Bean yellow dwarf virus (BYDV) which infecting
dicotyledonous species. Their genome contains a large and small intergenic
region (LIR and SIR respectively) located at opposite sides of the viral
genome. Four proteins are considered to be encoded by the mastrevirus
genome: Rep A, Rep protein and movement protein (MP), capsid protein (CP),
are encoded by c-sense and v- sense strand respectively. The unique features
shared by all mastrevirus includes: (1) A ~80 nt long DNA sequence annealed
to a region within the SIR, present inside the viral particle, (2) expression of
two rep proteins: the full length Rep (translated from a spliced transcript of the
C1 and C2 ORFs) and Rep A (translated from C1 transcript).
2.2.15. Curtovirus (Beet curly top virus)
The type member is Beet curly top virus (BCTV). It includes leafhoppers-
transmitted viruses having monopartite genomes infecting dicot plants.
Besides MP and CP, their genome encodes V3 protein on the v-sense strand,
whereas c-sense strand encodes four open reading frames (ORFs): Rep, C2,
REn (replication enhancer) and C4.
2.2.16. Topocuvirus (Tomato pseudo curly top virus)
Recently, these are recognized as a distinct genus. The type member is Tomato
pseudo curly top virus (TPCTV). It is transmitted by tree-hoppers having a
monopartite genome. Genome study reveals that it is a natural recombinant of
2: Review of Literature
39
mastrevirus and Begomovirus (Briddon et al., 1996). Consistent with this
hypothesis, TPCTV can trans-complement the movement of DNA A
component of two bipartite Begomoviruses in the absence of their cognate
DNA B components (Briddon and Markham, 2001).
2.3. Minor Cucurbit Viruses
There are few viruses whose occurrence in cucurbits crops is not very
common, yet they have been reported in few cases and are not very widely
studied. Such viruses have been designated as minor viruses. Their brief
description is as follows.
2.3.1. Arabis mosaic virus (ArMV)
ArMV was described in cucumber in Europe (Van dorst and Van Hoof, 1965).
In seedlings, ArMV first causes yellowing, later leaf necrosis, growth
reduction and death (Hollings, 1963). Lateral axillary dwarfed shoots form in
ArMV infected plants that fail to bear fruits.
2.3.2. Tobacco ringspot virus (TRSV)
TRSV is mainly transmitted by nematodes (Xiphinema americanun). Melons
and cucumbers are most commonly affected by this virus. The virus has been
known on rare occasions to be seed borne in cucurbits. The newly infected
leaves show a very bright mosaic with plant stunting. but subsequent leaves
are reduced in size and develop a dark green color (Smith, 1957; Wingard,
1928).
2.3.3. Tomato ringspot virus (TmRSV)
TmRSV causes severe damage to summer and winter squash, but shows only
mild symptoms in the other cultivated cucurbits. Like TRSV, TmRSV is
2: Review of Literature
40
nematode transmitted and can be overwintered on many weed species without
expressing symptoms (Schmelzer, 1977).
2.3.4. Clover yellow vein virus (CYVV)
CYVV is an aphid- transmitted virus that can infect summer squash and was
previously considered to be the severe strain of bean yellow mosaic virus. The
virus produces a yellow specking on the foliage of infected plants (Zitter and
Banik, 1984).
Viruses are, thus, the most common causes of diseases affecting
cucurbits. These diseases result in losses through reduction in growth and
yield and are responsible for distortion and mottling of fruit, making the
product unmarketable. It is becoming obvious that: now, it is upon the crop
protectionists including plant pathologists and entomologists as well, to design
and formulate ways or mean to combat all enemies of the crops, so that the
growers (farmers) may try to minimize the losses caused by plant viruses to
the crops. The severity of individual virus diseases may vary with the locality,
the crop variety, and from one season to the next.
2.4. Selected Cucurbitaceous Crops and Their Virus Diseases Description of the four cucurbits crops studied under present investigation
with their viral disease is given as follows:
2.4.1. Momordica charantia (Bitter gourd) Commonly known as ‘bitter gourd’, somewhere it is also known as bitter
melon and belongs to family: Cucurbitaceae, Genus: Momordica, Species:
charantia. A total of 60 species are reported world wide and out of them seven
are available in India but only Momordica balasmina, M. charantia, M.
chochinchinesis, M. dioica are commonaly available and cultivated in one or
2: Review of Literature
41
other part of north eastern region (Yadav et al., 2004). M. charantia is totally
domestic, plant is monoecious, vine type and has variation in fruits shape and
size. Bitter melon is cultivated throughout eastern part of U. P. as a vegetable
food and medicine. It is a slender, climbing, annual vine with long stalked
leafs and yellow, solitary male and female flowers born in the leaf axils. The
fruit appears as a worthy gourd, usually oblong and resembling a small
cucumber.
Bitter gourd available in a variety of shapes and sizes. The typical
Chinese phenotype is 20 to 30 cm long, oblong with bluntly tapering ends and
pale green in color, with a gently undulating, warty surface. It is also a popular
vegetable in the cuisines of South Asia and the West Indies. In these culinary
traditions, it is often prepared with potatoes and served with yogurt on the side
to offset the bitterness, or used in sabji.
Bitter gourd (M. charantia) is regarded as one of the world’s major
vegetable crops and has great economic importance. It also is a promising
remedy that can help millions in the developing world who suffer from
metabolic disorders such as type-2 diabetes. These positive features may make
bitter gourd look like an all-purpose crop. Bitter melon has been used in
various Asian traditional medicine systems for a long time. Like most bitter-
tasting foods, bitter melon stimulates digestion. While this can be helpful in
people with sluggish digestion, dyspepsia, and constipation, it can sometimes
make heartburn and ulcers worse. The fact that bitter melon is also a
demulcent and at least mild inflammation modulator, however, means that it
rarely does have these negative effects, based on clinical experience and
traditional reports (Rao et al., 1999). The pantropical herbaceous vine M.
charantia is used in a traditional medicine wherever it is found. The bitter
melon or bitter gourd, is often used to treat diabetes (Halberstein and
Saunders, 1978; Mossa, 1985; Zhang, 1992; Arvigo and Balick, 1993). For
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this purpose, fruits are used in Asia (Zhang, 1992), while the above ground
part is used by the Maya in Belize (Arnason et al., 1981; Arvigo and Balick,
1993). Leaves, fruits and roots are also used to treat fever (Ayensu, 1978;
Halberstein and Saunders, 1978; Singh, 1986; Gir´on et al., 1991). They have
also been used in reproductive health as an abortifacient, birth control agent,
or to treat painful menstruation and to facilitate childbirth (Kerharo and Adam,
1974; West et al., 1981; Burkill, 1985; Mossa, 1985). While, the ethnobotany
of the plant in India and the Caribbean has been well documented
(Chakravarty, 1959; Morton, 1966; Wong, 1976; Halberstein and Saunders,
1978; Gir´on et al., 1991). The antiviral activity of M. charantia is one of the
important features of plant listed in literature (Anani et al., 2000; Hudson et
al., 2000).
Though, it has been claimed that bitter melon’s bitterness comes from
quinine, no evidence could be located supporting this claim. Bitter melon is
traditionally regarded by Asians, as well as Panamanians and Colombians, as
useful for preventing and treating malaria. Laboratory studies have confirmed
that various species of bitter melon have anti-malarial activity, though human
studies have not yet been published (Tan et al., 2008).
2.4.1.1. Virus diseases of M. charantia
Earlier in 1970, virus disease of bitter gourd was observed around Coimbatore,
India. The virus was transmitted by five species of aphid vector, the virus was
tentatively designated as Bitter gourd mosaic virus (Nagarajan and
Ramkrishanan, 1970). Later, the association of Begomovirus with bitter melon
was observed in Lucknow Uttar Pradesh, India. The symptoms consists of
upward curling, shortening and distortion of leaves, the infected bitter melon
fruits were stunted and deformed, disease incidence was high as 100%.
Whitefly (Bemisia tabaci) could transmit the associated virus from infected to
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43
healthy one (Khan et al., 2002). Natural occurrence of yellow mosaic disease
was observed on bitter melon, the infected plants further increased with the
severity of the symptoms and quality of fruits were reduced with compare to
healthy one (Raj et al., 2005b). A severe mosaic was observed on bitter gourd
crop during 1989 in western U. P. and association of an apparently
undescribed potyvirus was established with the severe mosaic disease of bitter
gourd. The isolate was identified as a strain of Watermelon mosaic virus-
1(Tomar and Jitendra, 2005). Bitter gourd yellow mosaic virus (BGYMV) is
one of the viruses affecting bitter gourd, transmitted by the whitefly, Bemisia
tabaci. A minimum of 5 white flies are required to transmit the virus
(Rajinimala et al., 2005). M. charantia is natural host of Papaya ring spot
virus (Ohtsu, 1988; Gonzalez et al., 2003; Chin and Ahmad, 2007), Zucchini
yellow mosaic virus-1 (Ohtsu, 1988; Fukumotu et al., 1993), Watermelon
silver mottle virus (Tokashiki and Yasuda, 1991), Cucumber mosaic virus
(Takami et al., 2006), Squash vein yellowing virus and Cucurbit leaf crumble
virus (Adkin et al., 2008).
The association of Indian cassava mosaic virus (ICMV) with yellow
mosaic disease of bittergourd (M. charantia) has been reported from Tamil
Nadu, South India (Rajinimala and Rabindran, 2007).
Recently, Tomato leaf curl New Delhi virus infecting bitter gourd in
Pakistan was observed. Plants showing yellow blotch symptoms in several
fields, with an average incidence of 60-70% (Tahir and Haider, 2005) and in
Japan presence of Melon yellow spot virus on this crop was reported
(Shigeharu et al., 2009).
Literature revealed several reports of Begomovirus and other plant
viruses on bitter gourd from India (Mathew, 1991; Pandey and Joshi, 1991;
Khan et al., 2002; Rajinimala et al., 2005; Raj et al., 2005b; Tomar and
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44
Jitendra, 2005; Rajinimala and Rabindran, 2007) and abroad (Purcifull et al.,
1984; Tahir and Haider, 2005; Wall et al., 2006).
Presence of geminiviruse on M. charantia from the eastern region of
India was confirmed only by serological relationships (Raj et al., 2005b),
while at molecular level no reports are available from U. P. on this particular
important vegetable.
2.4.2. Trichosanthes dioica (Parwal)
Pointed gourd and commonly known as Parwal, Parmal, Parwar, Parora, Patal,
Patola etc, belongs to family Cucurbitaceae, genus Trichosanthes, sp. T.
dioica. It is one of the most nutritive cucurbit vegetable and holds a coveted
position in the Indian market during summer and rainy seasons. It is a
perennial crop, and is highly accepted due to its availability for eight months
(February-September) in a year (Kumar et al., 2003). T. dioica is
morphologically distinct from the other cucurbitaceous species due to its well-
established dioecism, perennial nature and vegetative means of propagation.
The genus Trichosanthes is considered to be of Indo-Malayan origin. It is a
large genus with 44 species, of which 22 are found in India. Although, the
Bengal-Assam area in India is considered as its primary center of origin, its
wild forms (T. japonica and T. multiloba) are found throughout the northern
India. A large number of local varieties are popular with the growers in their
respective areas.
Pointed gourd fruits are generally dark green, small, oval or light
green, long, pointed at both end without striped or black irregular striping,
round or regular striped having quite long or pale yellow are popular.
Pointed gourd is extensively grown in eastern parts of India (Uttar
Pradesh, Bihar, Assam and West Bengal). It is also cultivated in Orissa,
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Madhya Pradesh, Maharashtra and Gujarat. In India, this crop is cultivated in
about 14000 ha in Bihar and in ~10,000 ha in UP. Fruits of pointed gourd are
rich in protein and vitamin A (Sheshadri, 1990).
Pointed gourd has also high industrial value as different types of jam,
jelly and pickles can be made from this vegetable. T. dioica also known as
Kadu- padvala, is used in liver affections and jaundice. T. dioica was found to
possess anti-inflammatory activity, blood sugar, serum cholesterol, high
density lipoprotein, phospholipids and triglyceride lowering activity (Jaouad et
al., 2006). Phytochemical evaluations of aqueous and ethanolic extracts
consisted of saponins, tannins and a non-nitrogenous bitter glycoside
trichosanthin.
T. dioica is also known to have antiulcerous properties (Som et al.,
1993). The fruits and seeds have some prospects in the control of some cancer-
like conditions and haemagglutinating activities (Asolkar et al., 1992). Fruits
of T. dioica contain ample quantities of elements like Mg (9 mg), Na (2.6 mg),
K (83 mg), Cu (1.1 mg), S (17 mg) and Cl (4 mg): all the values are per 100 g
of edible part. Fruits are easily digestible and diuretic in nature (Som et al.,
1993; Chopra et al., 1956), In addition, fruit and other parts of the plant like
leaves and tuberous roots are used in the indigenous system of medicine
(Kirtikar and Basu, 1995).
According to Ayurveda, the plant is used for bronchitis, biliousness,
cancer, jaundice, liver affections, cough and blood disease. It is also used as
antipyretic diuretic and cardiotonic purposes (Kirtikar and Basu, 1975).
Pointed gourd is propagated by vine and root cuttings, layering and
tissue culture. October month is the best time for cutting when vines are
mature and fruiting is completed and harvesting is over (Kumar et al., 2003).
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2.4.2.1. Virus disease of Trichosanthes
Limited reports are available on virus disease of Trichosanthes dioica.
Watermelon mosaic virus was first reported virus on T. dioica in India in 1977
(Bhargava, 1977). Later, this crop was detected of virus infection in Florida, it
named as Trichosanthes virus (Purcifull et al., 1989). In 1996, report of
Potexvirus on Trichosanthes was made by Kucharek from Florida in 1996
(Kucharek et al., 1996). Since, then no report is available on occurrence of any
virus on Trichosanthes plant from any part of the world.
2.4.3. Cucurbita pepo (Pumpkin)
Cucurbita pepo (pumpkin) belongs to family Cucurbitaceae. The origin of
pumpkin is not known, although pumpkin is thought to have originated in
North America. The oldest evidence, pumpkin-related seeds dating between
7000 and 5500 B.C., were found in Mexico. Pumpkins are a squash-like fruit
that range in size (less than 1 pound to over 1000 pounds), shape, color, and
appearance (smooth or ribbed).
Since some squash share the same botanical classifications as
pumpkins, the names are frequently used interchangeably. In general,
pumpkins have stems which are firmer, more rigid, pricklier and are squarer in
shape than squash stems which are generally softer, more rounded, and more
flared where joined to the fruit. Pumpkins generally weigh 4–8 kg with the
largest (of the species C. maxima) capable of reaching a weight of over 34 kg.
The pumpkin varies greatly in shape, ranging from oblate through oblong. The
rind is smooth and usually lightly ribbed. Although pumpkins are usually
orange or yellow, some fruits are dark green, pale green, orange-yellow, white,
red and grey.
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Pumpkins are angiosperms, having both male and female flowers. The
color of pumpkins is derived from the orange pigments abundant in them. The
main nutrients are lutein, and both alpha- and beta- carotene. Their purpose is
to generate vitamin A in the body (Chakravarty, 1982).
The pumpkin plant has been known, since the dawn of time, in
country, and it is today widely cultivated as food and for decorative purpose,
in all warm and temperate parts of the globe. The seeds have been used in
traditional medicine as an anthelmintic and taenicide, demulcent, diuretic and
tonic. A tea made from the seeds has been used as a remedy for hypertrophy of
the prostate gland. Today the pumpkin seeds are utilized in the treatment of
urological symptoms associated with Benign Prostatic Hyperplasia (BPH).
The production of pharmaceutical products from the pumpkin seeds obviously
demands the use of a botanically defined specie, so as to obtain oils or extracts
that are reproducible from a chemically point of view (Madaus, 1979;
Bombardelli and Morazzoni, 1997).
The fruit can be cooked as a whole, topped, cored and stuffed. They
cook well in the microwave and make attractive individual servings. Mini
pumpkins are often used as attractive kitchen accessories for decoration before
they are converted into wholesome meals (Burgmans, 1994).
2.4.3.1. Virus disease of C. pepo
Viruses are the most important pathogens of cucurbits (cucumber,
watermelon, melon and pumpkins) belonging to the family Cucurbitaceae.
More than 30 infectious viruses causing destructive symptoms and
considerable economic losses were reported on these plants (Zitter et al.,
1996). Their occurrence, spreading, intensity of infection and destructiveness
depend on complex interrelations between the virus, its host plant, the vectors
and the environment. It is usually not easy to find appropriate control
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measures to reduce the extent of destruction. Viruses causing extensive losses
in cucurbit crops. Zucchini yellow mosaic virus known to be one of the most
destructive viruses of pumpkins (Dukic et al., 2001). Cucurbit viruses have
great economic importance in Yugoslavia (Stakia and Nikolia, 1966;
Pejainovski, 1978; Tosic et al., 1996).
Papaya ringspot virus - Type W (PRSV-W), Watermelon mosaic virus
2 (WMV2), Watermelon mosaic virus – Morocco (WMV-M), Zucchini yellow
mosaic virus (ZYMV) and Cucumber mosaic virus (CMV) can severely limit
the production of cucurbits (Lovisolo, 1980; Von wechmar et al., 1995).
WMV2, WMV-M, ZYMV and CMV have been recorded previously in South
Africa (Van der Meer, 1985; Van der Meer and Garnett, 1987; Von wechmar
et al., 1995). These viruses are transmitted in the stylet-borne manner by
aphids (Francki and Habili, 1990; Shukla et al., 1994). Tospovirus was
reported on C. pepo in Brazil (Bezerra et al., 1999). Aphids are an important
factor in the spread of viruses both within a field and over long distances. In
some cases, it has been noted that diseased plants seem to be more favorable
for rapid vector development than healthy plants (Swenson, 1968). Melon
necrotic spot virus is common in melon and cucumber crops worldwide and
can substantially reduce yields (Bos et al., 1984). Most natural hosts of
WmMV2 are in the gourd family, in addition to watermelon (Citrullus
lanatus), melon (C. melo), courggette (Cucurbita pepo), pumpkin (C. pepo)
and squash (C. maxima) (Purcifull et al., 1984). Squash mosaic virus occurs
naturally in C. pepo, primarily in squash, cucumber, courgette, melon and
watermelon. Infected plants may remain symptomless or show different
symptoms, such as mosaic, blister mottle, ring patterns, enations and leaf
deformations and in the case of early severe infections, the fruits are also
malformed (Campbell, 1971). Experimental infections have been recorded in
hosts in five families (Freitag, 1956).
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Pumpkin are natural hosts of Zucchini yellow mosaic virus, including
cucumber, courgette, melon, watermelon and zucchini squash. Symptoms in
infected plants vary, depending on virus strain and plant cultivars (Lecoq,
1981; Lesemann et al., 1983).
The crop was first reported to be affected by a mosaic disease in
northern India in early’s 1940s (Vasudeva and Lal, 1943) and subsequently
reported throughout India (Verma, 1955, Capoor and Ahmad 1975; Laths and
Gopalkrishnan, 1993; Singh et al., 2001). Literature revealed several reports
on C. pepo from India (Ghai et al., 1998; Tewari et al., 2001; Tripathi, 2005;
Singh et al., 2001, 2008) and from South Africa (Cradock et al., 2000,), Iran
(Farhangi et al., 2004), China (Chen et al., 2003), USA (Brust, 2000),
Germany (Riedle et al., 2002), Costa Rica (Hammond et al., 2005), Bulgaria
(Dikova and Hristova, 2002), Brazil (Moura et al., 2005), Thailand (Ito et al.,
2008), Yugoslavia (Dukia et al., 2001), Spain (Celix et al., 1996) and Egypt
(Abdel et al., 1998). Since, little attempt has been made so far to identify and
characterize viruses occurring on C. pepo in eastern U. P.
2.4.4. Luffa cylindrica (Sponge gourd)
Luffa cylindrica belongs to family Cucurtitaceae and was originated in South
Asia, South Europe and Africa. Luffa commonly called sponge gourd, loofah,
vegetable sponge, bath sponge or dish cloth gourd.
Sponge gourd is a sub-tropical plant, which requires warm summer
temperatures and long frost-free growing season when grown in temperate
regions. It is an annual climbing herb which produces fruit containing fibrous
vascular system. It is summer season vegetable. They have a long history of
cultivation in the tropical countries of Asia and Africa. Indo- Burma is
reported to be the center of diversity for sponge gourd. The main commercial
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production countries are China, Korea, India, Japan and Central America
(Joshi et al., 2004).
The fibrous vascular system inside the fruit after separating from the
skin, flesh and seeds, can be used as a bathroom sponge, as a component of
shock absorbers, as a sound proof linings, as a utensils cleaning sponge, as
packing materials, for making crafts as a filters in factories and as a part of
soles of shoes (Kirtikar and Basu, 1987). Immature fruit is used as vegetables,
which is good for diabetes. Oil is also extracted from seeds for industrial use.
The fruits are edible and eaten as vegetable. It is good for health. The seeds are
emetic and cathartic. Young fruits are cool, demulcent, productive of loss of
appetite and active of mind, bile and phlegm (Vashista, 1974). L. cylindrica is
used in medicine, make-up, slippers, hats and wallpaper (Young, 1989; Lee,
2003). Generally, L. cylindrica is cultivated from May to October in open
fields.
2.4.4.1. Virus disease of L. cylindrica
Virus disease have been considered to be one of the limiting factors affecting
cucurbit production, many reports are available for the presence of Zucchini
yellow mosaic virus, Papaya ring spot virus-W viruses on sponge gourd
(Chang et al., 1987; Hseu et al., 1985, 1987; Huang et al., 1989; Yeh et al.,
1988). L. cylindrica was found susceptible to Cucumber mosaic virus (Huang
et al., 1987). Earlier, in 1988 occurrence of Zucchini yellow mosaic virus was
reported from Japan (Somowiyarjo et al., 1988). Luffa virus disease was
observed in China in 2000 (Xu et al., 2000). L. cylindrica was more sensitive
to the infection of CMV and Zucchini yellow mosaic virus (ZYMV) in China
(Chen et al., 2003). Melon necrotic spot virus was reported on sponge gourd
from Japan (Matsuo et al., 1991). Recently, sponge gourd was found
alternative host of Cucurbit aphid-borne yellows (Xiang et al., 2008). In India,
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various disease on cultivated cucurbits like bitter melon, pumpkin, sponge
gourd and others have been reported from different regions (Vasudeva and
Lal, 1943; Capoor and Verma, 1948; Vasudeva et al., 1949; Bhargava and
Joshi, 1960; Reddy and Naraini, 1963; Hariharasubramanian and Badmi,
1964; Mitra and Naraini, 1965).
Sponge gourd (Luffa cylindrica), an important cucurbitaceous
vegetable in India, is affected by several virus diseases (Varma and Giri,
1998). It was found to be infected by Tomato leaf curl New Delhi virus in
India (Soharab et al., 2003). Later the association of Tomato leaf curl New
Delhi virus and Yellow mosaic virus was found (Soharab et al., 2006). Papaya
ring spot virus was detected in India for the first time in our country on this
important vegetable crop (Verma et al., 2006).
2.5. Methods for Identification and Characterization of Plant
Viruses A reliable and accurate detection of plant pathogen is a prerequisite to develop
disease management strategies. For diagnosis, symptomatology does not
provide a sound parameter and take longer time to establish the identity of
involved pathogen.
Plant viruses are generally identified by particle morphology, host
range and the serological properties of the coat protein. Cross-reactivity of
antisera raised against viruses from different groups has frequently been used
for classification and for the establishment of taxonomic relationships.
However, nucleic acid sequence data are accumulating rapidly and allow more
accurate relationships to be established between the individual members of
virus groups than serological methods do. Identification of a virus by
sequencing parts of its genome is often done if extensive serological analysis
cannot provide conclusive data about the nature of the virus. This approach
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requires the purification and isolation of the virus particles and the subsequent
cloning of parts of the virus genome. However, with novel molecular tools
like virus group specific PCR, sequence data from new viruses can be
obtained, even without the need to purify a virus or to clone parts of its
genome. Sequences obtained by such methods can reveal close and distant
relationships between new and existing viruses from a single group as can be
demonstrated for the group of Begomovirus which are the main group of plant
viruses occurring in cucurbitaceous crops.
Characterization of new Begomovirus appeared to be possible even in
case of mixed infections. For the identification of new Begomovirus several
general PCR primer sets have been developed which allow the amplification
of the complete genome of Begomovirus (Singh et al., 2008; Raj et al., 2010
Personal communication). For several other groups of plant viruses similar
PCR primer sets are available. Once identification of a virus has been
accomplished by nucleic acid sequencing, specific PCR primer sets can be
designed for very sensitive detection of the virus. However, to compete with
current serological methods for mass detection of plant viruses, the
development of fast and reliable PCR protocols including automated sample
preparation is a major challenge to overcome.
Alternatively, the molecular approach provides new possibilities to the
development of very specific and sensitive antisera against individual viruses
or (sub) groups of viruses. The expression of parts of a virus coat protein as
recombinant fusion products can provide insight in the location of epitopes on
the coat protein's amino acid sequence recognized by existing virus specific
and virus (sub)group specific antisera.
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2.5.1. Serological Methods
Serology is an indispensable tool for detecting viruses in infected plant
materials and also for the identity of the causal virus. Serological methods
were first applied to plant viruses by Dvorak (1927). Beale (1928) was the
first to establish the specificity of the serological reactions of plant viruses.
Plants containing different viruses also contained different specific antigens,
and this property of the viruses differentiate them from other members of the
group and provides valuable information on the virus identification.
Serology also known as the doubly antibodies sandwich-ELISA
method (DAS-ELISA) in some cases is not suitable for Begomoviruses
characterization, because it is difficult to prepare sufficient antiserum and it
lacks sufficient specificity (Brown et al., 2001). Serological test showed that
all Begomoviruses are related but there is a group of epitopes unique to
geminiviruses, which infect crops in the Old world and distinct set of epitopes
shared by Begomoviruses that infect crops in the New world (Thomas et al.,
1986). Serological methods used in detection of plant viruses are described
below.
2.5.1.1. Enzyme-linked Immunosorbent Assay (ELISA)
Since, the development of the microplate methods of ‘enzyme linked immuno
sorbent assay’ (ELISA) (Clark and Adams, 1977), ELISA has become a
routine method for plant virus detection and quantification of virus level in the
plant host. ELISA is simple to perform, sensitive, and easily used in large-
scale analysis. However, ELISA interpretation and sensitivity is improved by
the use of specialized equipment, and the development of viral specific
antibodies (Crespi et al., 1991; Al-Bitar and Luisoni, 1995)
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In “direct” ELISA procedures, the antibodies (usually as an immuno-γ-
globulin or IgG fraction of the antiserum) bound to the well surface of the
microtitre plate capture the virus in the test sample. The captured virus is then
detected by incubation with an antibody-enzyme conjugate followed by
addition of color development reagents (substrate or substrate/dye
combination). The capturing and detecting antibodies can be the same or from
different sources. Since, the virus is sandwiched between two antibody
molecules, this method is called the double antibody sandwich (DAS) ELISA.
In practice, DAS-ELISA is highly strain-specific and requires each detecting
antibody to be conjugated to an enzyme. There are several alternative
“indirect” forms of ELISA available for virus detection. In these methods,
antibodies raised in two different animal species and alternative ways of
immobilizing the virus in the wells of the ELISA plate have been used. One
approach, known as direct antigen-coating (DAC), antigen-coated plate
(ACP), or platetrapped antigen (PTA) ELISA, is to allow the virus, in the
absence of any specific virus trapping layer as in DAS-ELISA, to adsorb on
the plate surface by adding the test sample directly to the wells. In the second
step, virus antibody (usually called primary antibody) is added either as IgG or
crude antiserum. The primary antibody is then detected with anti species
antibodies (secondary or detecting antibody) conjugated to an enzyme,
followed by addition of color development reagents. The detecting antibody
binds specifically to the primary antibody since the former is produced against
IgGs from the animal in which virus antibodies are raised (e.g., if virus
antibodies are produced in rabbits, anti rabbit IgGs are produced in a second
species such as goats). It has certain disadvantages such as competition
between plant sap and virus particles for sites on the plate and high
background reactions.
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A second widely used approach is triple antibody sandwich (TAS)
ELISA. This is similar to DAS-ELISA, except that an additional step is
involved before adding detecting antibody–enzyme conjugate. In this step, a
monoclonal antibody (MAb), produced in another animal (usually mice)
different from the trapping antibody, is used. This MAb is then detected by
adding an enzyme-conjugated species-specific antibody (e.g., rabbit antimouse
IgG) that does not react with the trapping antibody, followed by color
development reagents. In the third, called protein A-sandwich (PAS) ELISA,
the microtitre wells are usually coated with protein A before the addition of
trapping antibody. The protein A keeps the subsequently added antibodies in a
specific orientation by binding to the Fc region so that the F (ab’)2 portion of
the antibodies traps virus particles. This can often increase the sensitivity of
the ELISA by increasing the proportion of appropriately aligned antibody
molecules. The trapped virus is then detected by an additional aliquot of
antibody which in turn is detected by enzyme-conjugated protein A and
subsequently color development reagents. Thus, in this method the antibody–
virus–antibody layers are sandwiched between two layers of protein A. As a
result, different orientations of the IgG in the trapping and detecting layers of
antibodies enable the protein A to conjugate to discriminate between them.
This permits use of unfractionated antisera. Thus, in indirect ELISA
procedures, the virus is detected by using a heterologous antibody conjugate
that is not virus-specific, but specific for the virus antibody or primary
antibody. As a result, a single antibody-conjugate can be used in indirect
assays to detect a wide range of viruses. Indirect ELISA procedures are more
economical and therefore suitable for virus detection in a range of situations
that include disease surveys and quarantine programs.
When ELISA was compared to molecular detection methods based on
DNA probes, it was found that ELISA was effective in detecting Begomovirus
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56
in purified preparations, but not in rude extracts of Begomovirus- infected
plants. In the latter case, the absorbance ratio of infected over healthy
(background) tissue was often too low to be considered reliable (Noris et al.,
1994). When ELISA was tested as a screening tool in a Tomato yellow leaf
curl virus-resistance breeding programme, it allowed an accurate
differentiation between susceptible and highly resistant genotypes. However,
ELISA also exhibited low sensitivity, which precluded degrees of resistance.
Several reports of viruses on cucurbitaceous crops through serological
assays are available in the literature (Purcifull et al., 1984; Zitter et al., 1996;
Sohrab et al., 2003; Tomar and Jitendra, 2005; Papayannis et al., 2005; Khan
et al., 2002; Orita et al., 2007; Rajnimala et al., 2009).
2.5.1.2. Dot Immunobinding Assay (DIBA)
It is a simple dot-blot immunoassay and the principle of DIBA are nearly the
same as those of ELISA, differing only in that the antigen or antibody is
bound to a nitrocellulose membrane instead of a microplate, and that the
product of the enzyme reaction is soluble. It has also referred as “DOT-
ELISA” i.e., ELISA on membranes. It is less expensive and labour-intensive
than conventional ELISA procedures and has been used to detect a variety of
plant viruses including membranes of different virus groups. DIBA has been
used to detect TYCLV in a number of studies with satisfactory results (Noris
et al., 1994). Moreover, DIBA has been used to screen for TYCLV- resistant
tomato genotypes. Unlike ELISA, DIBA was sensitive enough to allow a
differentiation among genotypes with varying degrees of resistance to
TYCLV. This differentiation was independent of the antibodies used.
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2.5.1.3. Tissue Blot Immunoassay (TBI)
Tissue blotting is a process of transfer of viral antigens from a freshly cut plant
tissue surface to nitrocellulose membranes. It is similar to, but not as
complicated as that of protein transfer from gels to nitrocellulose matrix. The
TBI technique also provides simplicity, rapidity and convenience for the assay
of a large no. of samples. The tissue blotting technique allows exact
localization of plant pathogens antigens in plant tissues. Direct tissue blot
immune assay is transfer of antigen from the specimens on to a nitrocellulose
membrane support by means of blotting a freshly cut tissue surface on to the
supporting substrate, followed by detection of antigens immobilized on the
membrane by the enzyme-tagged antibodies. It may not always reach the same
sensitivity as ELISA and DIBA. It can be performed with little equipment.
2.5.1.4. Immunosorbent electron microscopy (ISEM)
This is used for detecting antigens, or in ultrathin sections of virus infected
tissues. When the virus is suspended, the positive reaction can be identified
with the help of an electron microscope. Derrick (1973) introduced a method,
serologically specific electron microscopy, in which electron microscope grids
are coated with antibodies for the specific trapping of viruses. This method
subsequently came to be known as solid-phase immune-electron microscopy
(SPIEM) because one of the reactants (antibody) is first adsorbed to a solid
phase. Coating of the grid with a virus followed by treatment with an antibody
is not recommended, for it may cause some aggregation because of specific
migration on the grid. This method has been renamed immunosorbent electron
microscopy (ISEM) in 1982. Many variants of the ISEM method are in use,
and important of them include the following: Aggregation of viral particles,
Antigen Coating Grid Method, and Protein A- Coated grid method.
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2.5.2. Characterization Based on Viral Nucleic Acids Widespread application of immunological assays (serological methods) using
polyclonal antisera has served for decades for identification and classification
of viruses. They are based on the antigenic properties of the virus coat protein,
which represents only about 10% of the total virus genome and thus does not
take into account the rest of the virus genome. Further, variability in antiserum
produced globally has ambiguous results with the same antigen leading to
confusion and disagreements among the researchers. However, breakthrough
of hybridoma technology has offered significant advancement in immunology
and overcome many problems associated with polyclonal antisera. It has the
potential for producing an unlimited quantity of monospecific antibody ideal
for detection and characterization of plant viruses.
Nucleic acid-based detection methods (PCR based or hybridization
based), on the other hand, have the advantage that any region of a viral
genome can be targeted to develop the diagnostic test. In addition, there are
situations where immunological procedures have limited application in
particular for the detection of viroids.
2.5.2.1. Detection of the causal virus through Polymerase Chain
Reaction
Over the past decade, biotechnology and molecular biology has brought
radical change in virus detection technology. Recently, polymerase chain
reaction (PCR), the most powerful detection technique, has enabled the
amplification of extremely low number of viral RNA/DNA molecules. Due to
its sensitivity and versatility it has become a worldwide researchers tools in
plant virology and other area of biology.
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Today, PCR is a routine assay for amplification of part or all of the
Begomovirus genome, with a large array of Begomovirus specific primers
available (Navot et al., 1989; Mehta et al., 1994; Polston et al., 2001; Bosco et
al., 2004; Singh et al., 2008). Due to the very high sensitivity of the PCR
method, it has been used successfully to detect viral DNA in individual
whitefly adults (Navot et al., 1989), as well as in individual whiteflies in
various development stages (Polston et al., 2001).
PCR technique, developed by Kary Mullis in 1985, generates
microgram quantities of DNA copies (up to billion) of the desired DNA (or
RNA) segment, present even as a single copy in the initial preparation, in a
matter of few hours. The PCR process has been completely automated and
compact thermal cyclers are available in the market. The PCR is carried out in
vitro and utilizes the following: (1) DNA preparation containing the desired
segment to be amplified. (2) Two nucleotide primers, forward and reverse
(about 20 bases long). (3) The four deoxynucleoside triphosphate, viz., dTTP
(thymidine triphosphate), dCTP (deoxycyctidine triphosphate), dATP
(deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate) and
heat stable DNA polymerase e.g. Taq (isolated from the Thermus acquaticus).
Above procedure is applicable directly to DNA plant viruses (caulimo,
gemini, and badnaviruses); however, for diagnosis of plant viruses with RNA
genomes, the RNA target has to be “converted” to a complementary DNA
(cDNA) copy by reverse-transcription before PCR is begun. The cDNA
provides a suitable DNA target for subsequent amplification. During the
initial cycles of PCR, a complementary strand of DNA will be synthesized
from the cDNA template, and thereafter the reaction will proceed as for
double-stranded DNA described above. This process of amplification is called
reverse transcription-polymerase chain reaction (RT-PCR). On completion of
the reaction, the amplified DNA can be analyzed by agarose gel
electrophoresis. Many reports are available form India and abroad on
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detection of viruses on cucurbitaceous crops through PCR technique (Tahir
and Haider, 2005; Raj et al., 2005b; Singh et al., 2008; Shigeharu et al.,
2009; Rajnimala et al., 2009)
Besides, PCR techniques, nucleic acid hybridization approach is also
used for detection of plant virus.
2.5.2.2. Detection through Nucleic Acid Hybridization assays
The affinity of one strand of DNA for its complementary sequence is one of
the strongest and most exquisitely specific interactions found in nature. This
specificity has been exploited in developing nucleic acid hybridization assays,
which are based on the homology between two strands of nucleic acid
(DNA:DNA, DNA:RNA or RNA: RNA). In these assays, a single-stranded
complementary nucleic acid (either DNA or RNA), which has been “labelled”
with a reporter molecule is used as a probe to form a hybrid with the target
nucleic acid. The double-stranded probe-target hybrid molecules are then
detected by several methods, depending on the reporter molecule used. The
dot- or spot-blot hybridization assay is a commonly used technique in plant
virus diagnostics. The whole process involves solid–liquid hybridization,
wherein (i) the target nucleic acid (i.e., viral nucleic acid in the sample to be
tested) is spotted and immobilized onto nitrocellulose or positively charged
nylon membrane, (ii) the free binding sites on the membrane are blocked with
a nonhomologous DNA (usually salmon sperm or calf-thymus DNA) or
protein (usually bovine serum albumin or nonfat dried milk), (iii)
hybridization is allowed to take place between the bound viral nucleic acid and
the probe (which is free in the hybridization solution), (iv) the nonhybridized
probe is removed from the membrane by a series of washing steps at defi ned
stringency, and (v) the target sequences are assayed by detecting the reporter
molecule in the hybridized probe. Complementary DNA (cDNA) clones,
specific to any region of the viral genome, are commonly used as a probe to
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detect virus in plant extracts. To produce cDNA clones, the viral RNA is
usually converted to double-stranded DNA and cloned into suitable vectors
(Sambrook et al., 1989). The major advantages of using cloned DNA are
purity and unlimited supply of the probe. In addition, cloning of DNA into
vectors immortalizes the cDNA, so that such clones are available for use at
any time and can be supplied to different labs for use in virus diagnostics,
thereby offering uniform test results. The choice of labelling method is
dictated by the nature of the probe to be used.
DNA probes may be generated by nick translation, random primed
labelling, and by polymerase chain reaction (PCR), whereas RNA probes are
prepared by in vitro transcription. Unlike DNA probes, single-stranded RNA
probes can hybridize only with the target sequence without re-annealing and
RNA:RNA hybrids are more stable than DNA:RNA hybrids. However, the
potential risk of degradation of RNA probes due to RNAase contamination
during hybridization and high costs of generating such probes make the use of
DNA probes more common in virus detection assays. Khan et al. (2002) and
Sohrab et al. (2003) used this technique to detect the yellow mosaic disease of
cucurbitaceous crops.
2.5.3. Advantage of serological and molecular characterization of viruses on cucurbitaceous crops
In order to improve the productivity of cucurbitaceous crops and minimize the
infection of this economically important plant, it’s proper diagnosis and
control is essential. During the last two decade much advances has been made
in diagnostic for the detection of viral diseases, but incidence of viral infection
is increasing very fast. Serology is a traditional technique for virus detection,
based on antigen-antibody interaction. Molecular biology technique also play
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the major role to detect and characterize the viruses. These techniques have
shown great potential as far as specificity and sensitivity are concerned.
Cucurbitaceous crops are one of the most sensitive commodity areas of
research in India today. As we know the importance of Cucurbitaceous crops
and also about the wide spread of viruses on it, as a initial step towards
resolving these constraints, there is a need to develop a sound research
strategy and programme, and to achieve the above objective, serology is the
best way for detection, and molecular technique is the way to properly
identify and characterized the viral infection. On the basis of morphology and
symptomatology some time it’s difficult to identifying the viruses, and in the
case of mixed infection, it’s almost impossible. When the identification and
characterization is achieved, it would be easier to find suitable management
strategies to control the infection and keep the plant healthy. So, studies
through serological and molecular technique would generate important
information towards management and check of further spread of the detected
viral disease.