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

2: Review of Literature

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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


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-


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,


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).


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 Vadodara AF413671 ToLCGV-[IN:Vad:99]


Tomato Varanasi AY190290 AY190291

ToLCGV-[IN:Var:01]


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-


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]


Cotton Hissar EF063145 ToLCNDV-IN[IN:His:Cot:05]


Tomato Lucknow X89653 ToLCNDV-IN[IN:Luc]


Potato Meerut AY286316 AY158080

ToLCNDV IN[ IN:Mee:Po12:02]


Tomato New Delhi DQ169056 DQ169057

ToLCNDV-IN[IN:ND:05]


- New Delhi AY428769 AY438563

ToLCNDV-IN[IN:ND:AVT1]


- New Delhi U15016 ToLCNDV-IN[IN:ND:Mld:92]


- New Delhi U15015 U15017

ToLCNDV-IN[IN:ND:Svr:92]


Luffa Sonepat AY939926 AY939924

ToLCNDVIN[ PK:Dar:T5/6:01]


- Pakistan AF448058 AY150305

ToLCNDVIN[ PK:Dar:T5/6:01]


- Pakistan AF448059 AY150304

ToLCNDV-IN[PK:Isl:T1/8:00]


Chili Pakistan DQ116880 DQ116882

ToLCNDV-IN[PK:Kha:Chi:04]


Luffa Pakistan AM292302 ToLCNDV-IN[PK:Mul:Luf:04]


Solanum nigrum

Pakistan AJ620187 AJ620188

ToLCNDV-IN[PK:Sn:97]


Solanum nigrum

Pakistan DQ116885 ToLCNDV-IN[PK:Sol:04]


Solanum nigrum

Pakistan DQ116883 ToLCNDVIN[ PK:Sol:PT10:04]


- 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]


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


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.


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,


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).


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


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).


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.


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


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


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


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


42

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


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


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,


45

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).


46

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.


47

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


48

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).


49

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


50

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,


51

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


52

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.


53

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)


54

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.


55

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


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.


57

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.


58

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.

2. review of literature - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/30692/9/09...2: review...

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