Chapter 2 Review of Literature

6

Watermelon

The species Citrullus lanatus (watermelon) belong to Cucurbitaceae family, which consists of

nearly 100 genera and over 750 species (Yamaguchi, 1983). They are widely distributed in the

tropics and subtropics, and a few species occur in temperate region. Watermelon grows well in

alluvial and sandy soils even in arid regions and coastal saline areas. In the gangatic plains, early

sowing is done in November and extended up to February; in South and Central India

watermelon is grown almost throughout the year.

Watermelon is a major cucurbit crop that accounts for 6.8% of the world area (second

behind tomato) devoted to vegetable production in 2005. A rough estimate of annual world value

of watermelon exceeds $ 15 billion. As per an estimate, the total production of cucumber, melon

and watermelon has increased more than fourfolds in the last 40 years (FAO, 2006) (Fig. 2.1).

Watermelon is the most popular cucurbits, followed by cucumber, and melon (FAO, 2005).

Watermelon is originally from Africa and grown in more than 96 countries worldwide. China is

the world leader in watermelon production with 70.3% of total production in 2005. Other leading

countries are Turkey (4.7%), Iran (2.3%), United States (2.2%) and Egypt (1.7%). India (290,485

mt) occupies 26th position in watermelon production (FAO, 2005).

Watermelon is an economically important fruit crop and valuable alternative source of

water in desert areas. It is a good source of lycopene, citrulline and important minerals and

vitamins. It has the highest lycopene content among fresh fruits and vegetables; watermelon

contains 60% more lycopene than tomato. Lycopene in the human diet is associated with

prevention of heart attacks and certain cancers. Rind of watermelon contains an important natural

compound called citrulline, an amino acid that the human body makes from food. Citrulline is

found in high concentration in liver, and is involved with athletic ability and functioning of the

immune system (Perkins-Veazie et al., 2001). It is a good source of fiber which is important for

keeping digestive tract operating properly by preventing constipation, hemorrhoids and

diverticular disease.

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7

Watermelon Diseases

Watermelons are susceptible to several diseases that attack the roots, foliage and fruit.

The most common diseases of watermelon are anthracnose, fusarium wilt, downy mildew,

gummy stem blight (fungal diseases), root-knot nematode and virus diseases (NC State

Watermelon Disease Handbook). Around the world, over 10 viruses are known to be a problem

in watermelon production (Provvidenti, 1986). The major viruses affecting watermelon are

Papaya ringspot virus-watermelon strain (PRSV-W, formerly Watermelon mosaic virus-1),

Watermelon mosaic virus (WMV, formerly Watermelon mosaic virus-2), and Zucchini yellow

mosaic virus (ZYMV). All three viruses are non-persistently transmitted by many species of

aphids, and mixed infections are common (Adlerz and Crall, 1967; Mohr, 1986; Provvidenti,

1991; Wehner et al., 2001). An emerging destructive bud necrosis viral disease of watermelon

was first recorded in Taiwan in 1988 (Yeh et al., 1992) followed in southern India during 1991-

1992 (Singh et al., 1996), recognized as Watermelon bud necrosis virus (WBNV) (Jain et al.,

1998). In India, cultivation of watermelon is affected by many viral diseases: Cucumber green

mottle mosaic Virus (CGMMV), Cucumber mosaic virus (CMV), PRSV-W and WMV

(Bhargava et al., 1975; Vani and Varma, 1993).

Watermelon bud necrosis virus (WBNV)

Watermelon bud necrosis virus (WBNV) is an emerging Tospovirus, (family

Bunyaviridae) on cucurbits in India causing up to 100% crop loss in watermelon (Jain et al.,

2010). The affected plant produces a range of symptoms characterized by mottling and yellowing

of leaves, upright and stunting of vines and death of shoots due to necrosis of buds. The disease

is wide spread in watermelon growing areas in Karnataka, Andhra Pradesh and Maharashtra with

an estimated yield loss of 60-100% (Singh et al., 1996). The virus was initially identified as a

Tomato spotted wilt like virus under the genus Tospovirus, family Bunyaviridae based on virion

morphology, serology, host range and transmission by thrips, subsequently, the virus was

recognized as Watermelon bud necrosis virus (WBNV) based on the characteristics of

nucleocapsid protein gene sequence (Jain et al., 1998).

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8

The genus Tospovirus

Historical account

Bunyaviridae is the largest family of viruses, with over 200 species. This family

(Hantavirus, Nairovirus, Orthobunyavirus and Phlebovirus) largely infects vertebrates and there

are a small number of plant virus members, which form the genus Tospovirus which are

propagatively transmitted by thrips species, named after the type species Tomato spotted wilt

virus (TSWV) (Francki et al., 1991). The first report of the “spotted wilt” disease of tomato dates

back to 1915 when it was described this plant disease in Australia (Brittlebank et al., 1919).

Later it was demonstrated that the causal agent of the infectious disease was a virus (Samuel et

al., 1930) for which the name Tomato spotted wilt virus (TSWV) was coined. This taxon was

categorized as a monotypic virus group consisting of a single virus (TSWV) until the report of

Impatiens necrotic spot virus (INSV) after a long gap in 1987 (Law and Moyer, 1990). In 1992,

six more tospoviruses namely Groundnut bud necrosis virus (GBNV), Iris yellow spot virus

(IYSV), Melon yellow spot virus (MYSV), Peanut chlorotic fan virus (PCFV), Watermelon bud

necrosis virus (WBNV) and Watermelon silver mottle virus (WSMoV), were reported.

Groundnut ring spot virus (GRSV) and Tomato chlorotic fan spot virus (TCFV) were reported

soon after in 1993. In 1997, two more tospoviruses - Chrysanthemum stem necrosis virus

(CSNV) and Zucchini lethal chlorosis virus (ZLCV) were reported (Rezende et al., 1997). This

was followed by the identification of Peanut yellow spot virus (PYSV) in 1998 (Satyanarayana et

al., 1998), Capsicum chlorosis virus (CaCV) in 2002 (Lee et al., 2002; McMichael et al., 2002;

Knierim et al., 2006) and Chrysanthemum stem necrosis virus (CSNV) (Mumford et al, 2003) in

2004. Recently Tomato zonate leaf spot virus was identified from China (Dong et al., 2008). The

complete sequences of seven Tospovirus species (TSWV, INSV, GBNV, WSMoV, MYMV,

CaCV and TZSV) are available. Currently recognized tospoviruses are listed in Table 2.1.

Geographic distribution

Tospoviruses are distributed worldwide (Fig. 2.2). Of the 19 Tospovirus species recorded

worldwide, the Asian continent has by far the greatest diversity. TSWV is well-established in

most other production areas of the world and occurs in the Middle East and Iran, whereas in Asia

TSWV is only reported from a few countries, such as Japan, Korea, and Taiwan (Mumford et al.,

Review of literature

9

1996; Jones and Sharman, 2005). INSV occurs in the Middle East and South Asia where it

infects a range of crops (Lebas and Ochoa-Corona, 2007). Outbreaks of IYSV were reported in

Israel as early as 2000 (Kritzman et al., 2000) and the virus continues to be of concern for onion

and ornamental production in Israel. IYSV was first reported in onion in India in 2006 (Ravi et

al., 2006) and is now well-established in several western states. Besides TSWV and INSV

(Ghotbi et al., 2005), the identification and characterization of Tomato yellow ring virus (TYRV)

in Iran in potato, tomato, soybean, and ornamentals suggests its potential to affect the production

of some or all these crops in the future (Hassani-Mehraban et al., 2005; Winter et al., 2006;

Rasoulpour and Izadpanah, 2007; Golnaraghi et al., 2008). In India, tospoviruses are emerging

constrains in pulses, oilseeds and vegetables. So far, five distinct tospoviruses are reported from

India e.g., CaCV (Krishnareddy et al., 2008), Groundnut bud necrosis virus (GBNV)

(Satayanarayana et al., 1996), Peanut yellow spot virus (PYSV) (Satayanarayana et al., 1998),

Iris yellow spot virus (IYSV) (Ravi et al., 2006) and WBNV. Only GBNV and WBNV are most

prevalent in India affecting leguminous, solanaceous and cucurbitaceous crops (Jain et al., 2007).

Among tospoviruses, GBNV is currently recognized as the most economically important virus

negatively impacting the cultivation of several crops in parts of India, China, Nepal, Srilanka and

Thailand. Its distribution is restricted to Asian countries. Another important Tospovirus species,

WBNV continues to cause severe yield losses of up to 100% in various cucurbitaceous hosts in

India (Singh et al., 1996; Mandal et al., 2003; Jain et al., 2007; Kumar et al., 2010) (fig. 2.1).

More recently, CaCV was reported from Thailand and India (Premachandra et al., 2005, and

Krishnareddy et al., 2008) and in Taiwan (Chen et al., 2007). Chrysanthemum stem necrosis

virus (CSNV) was reported from Japan (Matsuura et al., 2007).

Economic impact of Tospoviruses

Tospoviruses are infecting a broad range of vegetables, agronomic crops and ornamentals

worldwide. They are cosmopolitan and infect more than 1000 plants species spanning

dicotyledonous and monocotyledonous plants. It has been estimated that tospoviruses cause

global yield loss upto $1 billion in a wide range of crops. TSWV causes serious losses in the

yield of groundnut and many other crops in Australia, India, Nepal, China, Thailand and USA

Review of literature

10

(Reddy, 1995). TSWV among the top ten of the most damaging plant viruses in the world. In

1991 TSWV in tomato recorded a loss of 1.26 t in fruit yield per hectare due to infection.

Due to GBNV infection estimated Crop loss upto US $ 89 million per year in India from

1976- 86. During 1993, GBNV caused 70-90 % loss of groundnut yield at Mainpuri, India.

Besides groundnut, losses up to 29 % have been recorded in potato due to stem necrosis disease

caused by GBNV (Singh et al., 1997). In Thailand also, GBNV causing bud necrosis disease in

groundnut is ranked first in economic importance, because of severity and widespread

distribution (Wongkaew, 1995).

Yield of watermelon and melon can be significantly reduced when a plant becomes

infected with WSMoV, but no precise figures are currently available. Iwaki et al. (1984)

described considerable reductions in yield and quality of infected watermelon in Japan, but there

is no more detail information. Yeh et al. (1992) described reduced fruit set in affected

watermelon plants in Taiwan and the fruit that developed was small and malformed. They

considered the virus to be widespread throughout Taiwan and one of the major limiting factors

for melon and watermelon production. Singh and Krishnareddy (1996) described TSWV-W

infection of watermelon in India as causing damage of up to 100% when early infection

occurred. Boiteaux et al. (1995) speculated that the new disease of melon in Brazil may be

severe in commercial melon plantings especially as the natural population of thrips vectors

increases dramatically in central Brazil during the dry season.

Host range

TSWV has a host range spanning several hundred species in both monocotyledonous and

dicotyledonous plants. However, the TSWV host range is not typical of all tospoviruses. Most of

which have moderate or small host ranges. Although host ranges tend to vary from virus to virus.

Nicotiana benthamiana is a good assay host for most tospoviruses. Symptoms range from

classical chlorotic spots and concentric ring to veinal necrosis in leaves, stems, petioles and

flower petals (Moyer, 1999).

Host range of has been reviewed by various authors (Hausbeck et al., 1992; Van et al.,

1993; Gognalons et al., 1996; Verhoeven and Roenhort, 1998 and Chazivassiliou et al., 1999).

WBNV is serologically moderately related to Indian GBNV, Host range is very much different

Review of literature

11

from GBNV (Singh and Krishnareddy, 1996). WBNV infects the members belonging to the

families namely Amaranthaceae, Asteraceae, Chenopodiaceae, Cucurbitaceae, Fabaceae and

Solanaceae under glass house conditions (Singh and Krishnareddy, 1996).

Transmission

Tospoviruses are spread by small insects called thrips (Thysanoptera: thripidae) (Amin et

al., 1981; Cho et al., 1988). Less than ten species of thrips have been confirmed as vectors

of tospoviruses and specificity between thrips and Tospovirus species has been shown (Table

2.2). Tospoviruses replicate in their thrips vectors, thus the insects not only spread the virus, but

serve as a virus host. Thrips cannot transmit unless they acquire the virus during their immature

stages (Fig. 2.4). When larvae feed on infected plants, ingested virus crosses the midgut barrier

and enters the salivary glands. Transmission then occurs when the virus moves into the plant

with the saliva during feeding. A midgut barrier in adults prevents virus ingested during this

stage from moving to the salivary gland. While the insects remain infective for life, there is no

evidence of transovarial passage from one generation of thrips to the next. Seed transmission

of tospoviruses is not known to occur, but some of these viruses are commonly spread in infected

propagation material when crops are vegetatively propagated.

Detection and Diagnosis

Symptomotology

Susceptible host species and the symptoms generated by TSWV (Plant Viruses online

http://image.fs.uidaho.edu/vide/; Van de Wetering et al., 1996; Gera et al., 2000), INSV (Brunt

et al., 1996) and WSMoV (Yeh et al., 1996) are well documented. Tospovirus often mimic

symptoms caused by other problems such as nutrient deficiencies. The characteristics symptoms

of Tospovirus infections induced in floral crops are: brown, black, or white spots; necrosis on the

leaf petiole; yellow mottling or variegation; death of young plants or death of terminal meristems

of older plants; stunting; brown or black cankers on the stem; veinal necrosis; concentric ring

spots; mosaic and line or zonal patterns (Daugherty and Casey, 1998). Among different

Tospovirus species symptoms of WBNV in watermelon were described by Singh and

Krishnareddy (1996) and Kumar et al., (2010). Initial symptoms appear on young quadrifoliate

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Table2.2: Thrips species transmitting Tospoviruses

1: Capsicum chlorosis virus (CaCV); 2:Calla lily chlorotic spot virus (CCSV); 3:

Chrysanthemum stem necrosis virus (CSNV); 4:Groundnut bud necrosis virus (GBNV); 5:

Groundnut ring spot virus (GRSV); 6: Impatiens necrotic spot virus (INSV); 7: Iris yellow spot

virus (IYSV); 8: Melon yellow spot virus (MYSV); 9: Peanut chlorotic fan virus (PCFV); 10:

Peanut yellow spot virus (PYSV); 11: Tomato chlorotic spot virus (TCSV); 12: Tomato spotted

wilt virus (TSWV); 13: Tomato yellow t ring virus (TYRV); 14: Watermelon bud necrosis

virus (WBNV); 15: Watermelon silver mottle virus (WSMoV); 16: Zucchini lethal chlorosis

virus (ZLCV).

Species Common name Tospovirus

Frankliniella occidentalis Western flower thrips 3,5,6,11,12

Frankliniella chultzei Common blossom thrips or

cotton bud thrips

4,5,6,11,12

Frankliniella fusca Tobacco thrips 12

Frankliniella intosa - 5,6,11

Frankliniella bispinosa - 12

Frankliniella zucchini - 16

Thrips tabaci Onion thrips 7,12

Thrips setosus - 12

Thrips palmi Melon thrips 4,8,14,15

Scirtothrips dorsalis Chilli thrips 4,9,10

Ceratothripoides claratus - 1

Microcephalothrips

abdominalis

- 13

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13

leaves as mild chlorotic spots, mottling but in later quadrifoliate leaves develop in to necrotic and

older leaves became yellow with mottling symptom and infected plants died within a week. The

secondary symptoms like stunting, axillary shoot proliferation and malformation of leaflets were

noticed. Early infected plants were bushy, up straight and stunting. Necrosis of plants shown

from upper apical bud to downward of the plants. They are readily mechanically transmitted by

gentle rubbing of plants with inoculum prepared by grinding the infected samples in suitable

inoculation buffers. Indicator plants like N. benthamiana and cowpea cv. Pusa komal can be used

for identification of different tospoviruses.

Immunological methods

A turning point in the Tospovirus detection and diagnosis came with the production of

high quality polyclonal antisera against the nucleocapsid protein (N) and development of enzyme

-linked immunosorbent assay (ELISA) (Gonsalves and Truijillo, 1986). ELISA and various

serological blot assays using polyclonal or monoclonal antibodies are now the method of choice

for the diagnosis of tospoviruses in plants as well as thrips (Gonsalves et al., 1986; Cho et al.,

1988; Cho et al., 1989). Serology using antisera against the N protein has been used for the

classification of tospoviruses (Huguenot, 1990; de Avila, 1990). Polyclonal and monoclonal

antibodies have also been produced to other virion associated structural proteins (L, Gn/Gc) and

nonstructural proteins NSs and NSm. Attempts have been made therefore to develop a genus-

specific test. The availability of systems for expression of proteins in bacteria has facilitated the

production of antibodies to glycoproteins and the nonstructural proteins, which are otherwise

produced in low quantities and difficult to purify from other materials. The antiserum failed to

detect tospoviruses of the IV serogroup though it could detect the tospoviruses of serogroup I to

III (Heinze et al., 2000). Certain modifications that partly restored serogroup IV sequences

showed promising.

Nucleic acid based methods

In addition to serology and symptom expression, techniques based on detection of viral

specific nucleic acids are useful for diagnostic purposes. Riboprobes (labeled synthetic RNA

transcripts) and cDNA probes are available for diagnostic purposes (Huguenot et al., 1990).

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14

Eiras (2001) has developed dot-blot hybridization with digoxiin labeled probes for universal

detection of tospoviruses.

The reverse-transcription polymerase chain reaction (RT-PCR) using virus specific

primers is a powerful new technique for detecting small amounts of plant virus nucleic acids in

both plants as well as thrips (Jain et al., 1998). Recently multiplex RT-PCR has been reported for

the simultaneous detection and identification of two or more viruses (Nassuth et al., 2000;

Kuroda et al., 2002; Ito et al., 2002). Okuda and Handa (2001) developed a technique for the

simultaneous detection of five distinct Tospovirus species using degenerate primers and dsRNA

template by RT-PCR. Downstream primers were derived from the 3' untranslated sequences of S

RNA and upstream primers were derived from the degenerate sequences of the nucleocapsid

protein (N).

Morphology

Tospoviruses are quasispherical (Fig. 2.5) 80-110 nm in diameter and bound by a host-

derived membrane. Virions are composed of four proteins including the nucleocapsid (N)

protein, Gn and Gc glycoproteins and L (replicase) protein. Several copies of the replicase

protein are present in the virus particle. The L protein is assumed to serve the purpose of

replicase by analogy to other members of Bunyaviridae. The Gn/Gc proteins are components of

the membrane and form surface projections. The N proteins are tightly bound to the three RNA

species within the particles and form pseudo-circular nucleocapsid structures (German et al.,

1992; Moyer et al., 1999).

Genome organization

Tospoviruses have tripartite genome, consisting of three linear single stranded RNA

molecules, the large (L) (8.9kb), the medium (M) (4.8 kb) and the small (S)(2.9kb) RNAs. Some

copies of both polarities of S and M RNAs can be found in purified viral preparations (Moyer,

1999). Around 65-70nt at the 3' and 5' termini are complementary and the base pairing of which

lead to the pseudocircular nucleocapsid structure. Among these, the terminal nine nucleotides

(sequence 3’UCUCGUUAG) are strictly conserved among all RNA species of all the

tospoviruses (De Haan et al., 1989). The strategy used for the expression of individual genes in

tospoviruses is given in Fig. 2.6.

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15

L RNA

The L RNA is entirely of negative polarity, with an ORF located on the viral

complementary (vc) strand and codes for a protein with 2875 amino acids and a predicted

molecular mass of 331 kDa. This protein contains several nucleic acid polymerase motifs (De

Haan et al., 1991) including a highly conserved serine- aspartic acid (SDD) element found in all

the RdRp of all segmented negative strand RNA viruses (Tordo et al., 1992).

M RNA

The M RNA has ambisence coding strategy. It is 4.9 kb and encodes a 33.6 kDa

nonstructural protein (NSm) in the viral (v) sense and a 127.4-kDa precursor to the two viral

membrane glycoproteins, Gn and Gc in the viral complementary (vc) sense. The role of NSm in

cell- to-cell movement is supported by its early expression profile, ability to alter the size

exclusion limit of the plasmodesmata (Storms et al., 1998 b) and the observation that it forms

tubules, which is a feature of other movement proteins (Hull, 1991; Deom et al., 1992; Storms et

al., 1998a and b). The Gn/Gc precursor protein is expressed from sub genomic mRNA (3.5

kb).The glycoproteins also play a role in virion assembly.

S RNA

Like M RNA, the S RNA has an ambisense coding strategy. It is 2.9 kb long and codes

for the 52.4 kDa nonstructural protein (NSs) in the viral sense and the 29-kDa N protein the viral

complementary sense (De Haan et al., 1990). The NSs protein is expressed from a v sense 1.7kb

subgenomic RNA and the N protein from a 1.2kb vc sense subgenomic mRNA (De Haan et al.,

1990; Kormelink et al., 1992). The Tospovirus NSs protein has been shown to function in

suppression of RNA silencing during the plant infection cycle (Takeda et al., 2002; Bucher et al.,

2003). The NSs gene in some animal infecting members of Bunyaviridae play a role in virulence

of these viruses in their animal hosts which has led to the speculation that the NSs gene of the

tospoviruses may play a role in the thrips infection cycle (Vialat et al., 2000; Bridgen et al.,

2001). The nucleocapsid protein (N) plays a role in virus replication cycle in both structural and

regulatory manner. The N protein has been shown to form dimers in the absence of RNA (Kainz

et al., 2004; Uhrig et al., 1999) and to bind ss RNA but not dsRNA (Richmond et al., 1998).

Mutated forms of the N protein serve as inhibitors of virus replication (Rudolph et al., 2003). N

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16

and NSm genes of GBNV isolates from various hosts and locations have been sequenced from

India and the isolates were indistinguishable based on gene sequences. Sequence analysis of the

N genes of Indian GBNV isolates (Bhat et al., 2002, Jain et al., 2002, 2004, 2007; Thien et al.,

2003, Umamaheswaran et al., 2003) showed them to be highly conserved (96-99% amino acid

identity). Similarly the NSm gene from various hosts in family Fabaceae and Solanaceae was

924nt long and encoding 307 amino acids and shared amino acid identity of 98-100% among

them (Akram et al., 2004).

Management

For a long time, plant viruses have been controlled using conventional measures like

crop rotation, cross protection, discarding virus infected plants, using virus- free plants and

chemicals against virus vectors such as aphids, leafhoppers and thrips (Hull, 1991).

Conventional methods

Infected plants cannot be cured. Control measures aim to prevent or reduce the levels of disease

in crops by removing or avoiding sources of virus infection and minimizing spread by thrips.

Crop protection: (1) Old infected crops infested by thrips are a major source of virus and

should be sprayed for thrips and removed as soon as possible, particularly if young crops are to

be planted nearby. Avoid overlapping sowing of susceptible crops and sequential planting side

by side to minimize virus spread from one crop to the next. (2) Weeds along headlands, irrigation

channels and in fallow land provide host plants for thrips and tospoviruses. Disease levels are

often higher in crop rows adjacent to these areas. (3) Weeds that are flowering are particularly

attractive to thrips as they feed on pollen. Destroy weeds well before planting, not as crops are

planted, as virus infected thrips may migrate from the wilted weed to young plants. (4)

Maintaining a clean buffer zone free of weeds at least 25 m between a virus source and a

susceptible crop can considerably reduce virus levels.

Chemical Control: Reducing thrips population by use of insecticides (Table 2.3) can help

reduce virus spread. However, insecticides are often of limited value in Tospovirus control as

virus spread from non-crop areas (an important source of infection) and thrips only require

limited feeding times for virus transmission. Significant disease level can occur when thrips

numbers are low, and there is little evidence of direct feeding damage, as small number of thrips

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Table.2.3: Pesticides labeled for management of Thrips in greenhouses.

Class Common Name Trade Name

Insect growth regulator azadirachtin (neem) Aza-Direct, Azatin XL,

Neemix 4.5, Ornazin

Alkaloid nicotine Fulex Nicotine

Pyrethrins plus PBO pyrethrins and PBO

(piperonyl butoxide)

Pyrenone, 1100 Pyrethrum

TR, Pyrethrum TR, Pyreth-It

*Carbamate methiocarb Mesurol 75-W

Chloronicotinyl imidacloprid Marathon II, 1% Granular,

60 WP

Horticultural oil (paraffinic oil) horticultural oil Ultra-Fine Oil

Insect growth regulator: chitin

synthesis inhibitor

novaluron Pedestal

Insect growth regulator:

juvenile hormone analogue

kinoprene

fenoxycarb

Enstar II

Preclude

Microbial: macrocyclic lactone abamectin Avid 0.15EC

Microbial: entomopathogenic

fungus

Beauveria bassiana Botanigard ES, 22WP

Microbial: spinosyns spinosad Conserve SC

*Organophos-phates acephate 1300 Orthene TR, Orthene

TT&O, Orthene TT&O 97

*Organophos-phates and

Pyrethroid

chlorpyrifos and

cyfluthrin

Duraplex TR

Pyrethroid ester bifenthrin Talstar GH,N, Attain TR

Pyrethroid ester cyfluthrin Decathlon 20 WP

* Carbamates and organophosphates have the same mode of action and should be treated as the

same class.

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moving into a crop can result in significant virus transmission. Frequent use of insecticides may

also lead to development of insecticide resistance in thrips population.

Viruses can be introduced in infected seedling plants which then provide a virus source

throughout the life of the crop. Seedling production area should be located well away from

production areas, kept weed-free and systematically monitored for pests and diseases with a

regular spray schedule in place. Thrips-proof netting or UV absorbing plastic provides a higher

level of protection for seedling production.

Biological Controls: Biological control organisms currently under investigation

include: predacious mites such as Amblyseius cucumeris (also known as Neoseilus), Iphiseius

degenerans, Hypoaspis miles and Hypoaspis aculifer that feed on thrips larvae, predacious bugs

such as Orius insidiosus, entomophilic nematodes such as Thripinema nicklewoodii, and

entomopathogenic fungi, such as Beauveria bassiana, that attack all thrips life stages and kill in

two to 14 days. Use of biological control methods should only be implemented as part of a well-

planned IPM program.

The difficulties in the management of disease caused by tospoviruses are due to 1) The

broad host range of the virus, 2) The cosmopolitan nature of its vector(s), 3) Difficulties to

control these vectors chemically (Brødsgaad, 1994; Robb et al., 1995; Zhao et al., 1995) or

biologically (Morse and Hoddle, 2006) and, last but not least, the limited availability of

resistance genes for commercial breeding purposes.

Resistant varieties: So far, two single dominant TSWV resistance genes in host

plants have been well described. The first one has been identified in Lycopersicon peruvianum

for which high resistance levels to various TSWV isolates were observed. This gene (Sw-5) has

been introgressed into the tomato variety ‘Stevens’ that showed resistance against two additional

tospoviruses, GRSV and TCSV (Boiteux and Giordano, 1993). The Sw-5 resistance appears not

absolutely durable, as TSWV variants overcoming this resistance have already been reported

(Latham and Jones, 1998). The second resistance gene, denoted Tsw, was identified in Capsicum

chinense 'PI' accessions in which resistance displayed by a hypersensitive reaction (HR) to a

broad range of TSWV isolates. Like Sw-5, the resistance is governed by a single dominant gene

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(Black et al., 1991; Boiteux, 1995) and also for this trait resistance-breaking variants have been

identified (Roggero et al., 2002; Aramburu and Marti, 2003).

Non-conventional measures

The conventional methods for managing or controlling virus diseases have been

found to possess their own drawbacks. The cultural practices and chemical control of vectors are

not often satisfactory against many viral diseases (Varma, 1993). Natural resistance to the

viruses is limited or breeding programs have failed to produce cultivars with effective

resistance in a reasonable period of time extensive studies have been done on developing

engineered resistance (Goldbach and De Haan, 1993; Goldbach et al., 2003).

A plant with a new trait developed by transferring specific gene from any sources through

the method of genetic engineering is referred as ‘transgenic plants’. The transgenic plant

conferring resistance against pathogen is known as transgenic resistance. For developing virus

resistant transgenic plant (VRTP) the gene conferring resistance (transgene) can be derived from

virus, plant and microbes (Varma et al., 2002). Hamilton (1980) for the first time postulated that

pathogen it self may provide protection to plant diseases. Sanford and Johnson (1985) proposed

the theoretical concept of coat protein mediated resistance through genetic engineering methods.

The first practical demonstration of transgenic resistance in plant against virus was

demonstrated by Powell-Abel and co-worker in 1986 using tobacco and TMV system. Since

then, nucleotide sequences from several genes of plant viral genome such as coat protein gene,

replicase gene, movement protein gene, anti- sense RNA, defective interfering RNA and sat-

RNA, untraslated sequences have been found useful in developing virus resistant transgenic

plants (Praveen et al., 2004; Varma et al., 2002; Makesh kumar et al.,2002; Tabler et al., 1998;

Pappu et al., 1995; Lomonossoff, 1995; Grumet, 1994; Beachy, 1993; Golemboski et al., 1990;

Gerlach et al., 1987; Harrison et al., 1987). The coat protein (CP) mediated resistance showed

most promising approach against many different plant viruses. Studies have shown that

genetically engineered plant expressing a plant virus coat protein gene can resist infection by

homologous and in some cases hetrologous viruses (Namba et al., 1992; Beachy et al., 1990).

Recently, coat protein gene derived resistance has been successfully employed to develop virus

Review of literature

20

resistant crop varieties of cucurbits, papaya, potato and tomato, which showed promising

performance under practical field conditions.

Transgenic expression of the viral N protein rendered resistance to TSWV in tobacco

(Gielen et al., 1991; MacKenzie and Ellis 1992; Pang et al., 1992) and tomato plants (Ultzen et

al., 1995). In addition, expression of untranslatable N gene versions were shown to confer the

same level of resistance, indicating a pivotal role for the transgenically expressed viral RNA to

induce resistance, leading to the concept of RNA-mediated resistance (De Haan, 1992).

Further studies revealed that the resistance is based, like in many other cases of transgenic virus

resistance, on a process called post- transcriptional gene silencing (PTGS) or shortly, RNA

silencing (Baulcombe, 1996). A potential drawback of RNA-mediated virus resistance is its high

sequence specificity (Prins et al., 1996). As for N gene-mediated resistance, Jan et al. (2000)

showed that a minimum length of 236-387 base pairs of N transgene sequence is sufficient to

induce resistance or even shorter length (59-110 base pairs) provided that the latter sequence

is fused to a (non-target) carrier sequence e.g. green fluorescent protein (GFP).

Previous reports on transgenic TSWV-resistant plants using NSm gene sequences support

the view that tospoviruses are not silenced at the genomic level but at the transcript level.

Indeed the genomic RNA segments are fully encapsidated by N protein and seem less

attractive to serve as RNA silencing target. To obtain broad resistance against several

Tospovirus species, Bucher et al. (2006) designed an inverted-repeat construct (ds-construct)

containing short pieces of the N genes from 4 different tospoviruses which indeed provided

full resistance against all four viruses in Nicotiana benthamiana in more than 80% of the

transgenic lines tested. This kind of constructs is therefore particularly attractive for

transformation of plant species that suffers from low transformation/regeneration efficiencies.

Agrobacterium mediated plant transformation

Agrobacterium mediated transformation of plants has been found useful for the

improvement of economically important crops. Regeneration of plants under Solanaceae family

are relatively easy, therefore, the most of the genetic engineering studies are based on

solanaceous plants (Fraley et al., 1983; Herrera-Estrella et al., 1983). However, lack of suitable

regeneration protocol is a limiting factor in developing transgenic cucurbits (Wehner and Locy,

Review of literature

21

1981; Novak and Dolezelova, 1982; Malepszy and Nadolska-Orezyk, 1983). The most widely

used method of gene transfer in plant is through a disarmed Ti plasmid of Agrobacterium

tumefaciens (Zambryski et al., 1983) or through A. rhizogenes (Chilton et al., 1982). The first

successful transformation in cucurbit was achieved in cucumber, where plants were regenerated

from root tissues through A. rhizogenes containing a binary vector pARC8 (Trulson et al., 1986).

The major limitation of this approach was low frequency of regeneration from root tissues. Chee

(1990) used A. tumefaciens mediated transformation approach using cotyledonary tissues of

cucumber, which resulted in large number of transformed plants through somatic embryogenesis.

Fang and Grumet (1990) produced transgenic muskmelon plants by cocultivating cotyledonary

explant with A. tumefaciens strain LBA4404. Nishibayashi et al. (1996) produced transgenic

cucumber plant from hypocotyl explant inoculated with A. tumefaciens containing a binary

vector, pIG121-Hm. They had shown the effect of acetosyringone to enhance transformation

efficiency. Recently, Agrobacterium mediated transformation was reported in bottle gourd by

Han et al., (2005). They used Agrobacterium strain AGL1 carrying the binary vector

pCAMBIA3301 containing glufosinate ammonium resistance (bar) gene and -D-glucuronidase

(GUS) reporter gene for transformation of an inbred line of bottle gourd.

Management of virus in cucurbit by transgenic approach

There are many agricultural problems in the cultivation of the cucurbits for which

conventional breeding often does not have ready answers, and the current partial solution is the

increasing use of agrochemicals against pests and VR. In general, most cucurbit crops still lack

good VR, although some VR to some viruses and factors ameliorating disease responses are

available in all cucurbit species (Munger, 1993; Provvidenti, 1993). Major incompatibility

barriers between the important cucurbits crop species impede or prevent transfer of existing VR

from one species to another. Most cucurbit infecting viruses are insect vector or soil- borne, and

chemical control of the vector and/or soil sterilization is expensive, inefficient, and ecologically

damaging. Many problems of soil-borne pathogens in vegetables, including cucurbits, are now

being dealt with effectively by grafting onto resistant rootstocks (Lee and Oda, 2003). The

problems are difficult to solve through conventional measures, but solution can be found in

biotechnology. For example, melon fruit have been engineered for long shelf-life (Ayub et al.,

Review of literature

22

Table2.4. Comparison of Agrobacterium mediated transformation protocols of cucurbits

Kan: Kanamycin; Carb: Carbenicillin; AVG: L-(2-aminomethoxy-Vinyl) glycin; IAA: Indol-3

acetic acid; NAA: Napthalene acetic acid; 2,4-D: 2-4 dichlorophenoxy acetic acid; MS:

Musashige & Skoog; 2iP: N6-(2-isopentyl) adenine;

Crops Agrobacterium

strain

Vector Explant Co-cultivation

medium

Reference

Bottle

gourd

A. tumefaciens

(AGL1)

pCAMBIA3

301

Cotyledons MS+ 3mg/l BA+

0.5mg/l

AgNO3+0.5 mg/l

cefotaxim

Han et al.,

2005

Cucumber A. rhizogenes

(A4)

pARC8 Hpocotyl MSsalt +5M

NAA+2M BA

+ Kan 25 mg/l

Trulson et

al., 1998

A. tumefaciens

(C58Z707)

pGA482 Cotyledons MS salt +0.1

mg/l NAA+0.5

mg/l Kinetin+

100mg/lKan +

500 mg/l Carb

Chee et al.,

1990

A. tumefaciens

(EHA101)

pIG121-Hm Hpocotyl MS+ 1.1 mg/l

2,4-D+0.2mg/l

BA+1% Casine

hyrolysate+

500mg/l Carb+

50 or 100 mg/l

Kan

Nishibayas

hi et al.,

1996

Musk-

melon

A. tumefaciens

(LBA4404)

pCIBA10 Cotyledons MR medium+ 75

mg/l Kan+ 100

mg/l Cefotaxim

Fang et al.,

1990

Review of literature

23

1996; Flores et al., 2002). Most of the biotechnology of cucurbits has involved the production

of virus-resistant transgenic (VRT) crops.

Much cucurbit biotechnology has been directed towards melon and cucumber,

primarily because these are the economically most important cucurbit crops in the developed

world, with large public and private breeding programs. However, although much less work has

been published on Cucurbita species and watermelon (Table 2.4). There are following work

carried out on cucurbitaceous crop using biotechnology and their field trial (Table 2.5).

Cucumber: The CMV-resistant cucumber, 'Marketmore' series developed by

conventional breeding method was widely used by growers in the United States (Munger, 1993).

Gonsalves et al. (1992) developed transgenic cucumber plants using CP gene of CMV and

compared viral CP-derived resistance and host gene-derived resistance under field conditions

over three years. Transgenic cucumbers showed a long delay in symptom development with only

4-30% symptomatic plants nine weeks after transplanting, compared with 84% of

nontransformed controls and 13% 'Marketmore' resistant plants. ELISA of 12 wk post-planting

showed significantly fewer transgenic plants containing virus (28-41%) relative to both control

(85%) and commercial 'Marketmore' resistant plants (62%). Transgenic line T48 produced a

significantly higher yield (42% increases) than the other three transgenic lines analysed or the

nontransformed counterparts. Unlike resistance to CMV in tomato plants, CMV resistance in

commercial cucumber cultivars developed by conventional breeding is effective and readily

available. Therefore, CMV transgenic cucumbers are not recommended as replacement for the

resistant cultivars in the US.

Squash: Transgenic squash (Cucurbita pepo L.) lines expressing single or combinations

of CP gene constructs of CMV, ZYMV, and/or WMV 2 have been evaluated both in greenhouse

and field conditions.Three transgenic lines expressing CP genes of ZYMV and/or WMV 2 were

evaluated in the field under severe incidence of ZYMV and WMV 2 (Fuchs and Gonsalves,

1995). The transgenic line: ZW-20 was transformed with CP genes of both ZYMV and WMV 2,

line Z-33 CP gene of ZYMV, and line W-164 with CP gene of WMV2. Transgenic line ZW-20

showed excellent resistance to mixed infections by ZYMV and WMV 2 as no plant developed

severe symptoms, i.e. foliar mosaic, chlorosis, malformation or stunting. Only a few ZW-20

Review of literature

24

Table 2.5: Field trials of virus resistant transgenic crops developed using viral coat protein

(CP) gene as transgene.

Acronyms used in this table: -

CMV: Cucumber mosaic virus, PRSV: Papaya ring spot virus, PVX: Poty virus X, PVY: Poty

virus Y, TMV: Tobacco mosaic virus, ToMV:Tomato mosaic virus.; WMV2: Watermelon

virus2, WL: White leaf strain, ZYMV: Zucchini yellow mosaic virus, Ha: Hawaii, Thai:

Thailand.

Crop Source of CP gene Resistance to

virus/strain

Level of

field

resistance

References

Cucumber CMV CMV Moderate Gonsalves et al., 1992

Cantaloupe (CMV+ZYMV+W

MV2) CMV-WL

CMV, ZYMV,

WMV2

CMV

High

Moderate

Fuchs et al.,1997

Fuchs et al., 1997a

Papaya PRSV-[HA5-1] PRSV-[HA5-1] High Gonsalves et al., 1998?

Potato PVX PVX High Jongedijk et al., 1992

PVY-N PVY-N High Malnoe et al., 1994

PVY-N PVY-O Moderate Malnoe et al., 1994

PVY+PVX PVY, PVX High Kaniewaski et al., 1997

Squash ZYMV ZYMV

WMV2

High

No

Fuchs & Gonsalves,

1995

WMV2 WMV2

ZYMV

Low

Low

Fuchs & Gonsalves,

1995

ZYMV+WMV2 ZYMV, WMV2 High Fuchs & Gonsalves,

1995; Arce-Ochoa et

al., 1995

CMV+ZYMV+

WMV2

CMV, ZYMV,

WMV2

High Fuchs et al.,1998

Tomato CMV-WL CMV-Fyn High Fuchs et al., 1996

TMV-U1 TMV-U1 High Nelson et al., 1988

TMV-U1 TMV-PV230 High Sanders et al., 1992

TMV-U1 ToMV Weak Sanders et al., 1992

ToMV ToMV High Sanders et al., 1992

Review of literature

25

developed very mild leaf symptoms in the form of localized chlorotic dots or blotches. In

contrast, all plants of the transgenic lines Z-33 and W-164, and control plants developed severe

symptoms. Transgenic Z-33 was substantially infected (21%) by ZYMV and heavily infected

(98%) by WMV 2 after seven to 10 weeks. Both, transgenic W-164 and control plants were

totally infected with ZYMV and WMV2 after five weeks. Also ZW-20 fruits were symptomless

whereas all fruits from transgenic Z-33 and W-164, as well as from control plants were

unmarketable because of severe discoloration and distortion. The high resistance of transgenic

squash ZW-20 to infections by ZYMV and WMV 2 has been confirmed in several field tests at

different locations (Arce-Ochoa et al., 1995; Clough and Hamm, 1995). The low level of

resistance of W-164 appears to be due to a severe WMV 2 isolate which was able to severely

infect transgenic plants.

Cantaloupe: Cantaloupes (Cucumis melo L.) are severely affected by CMV, ZYMV,

WMV 2, and PRSV-W. Melon varieties with multiple resistances to these viruses would be

valuable to growers. Cantaloupes containing multiple viral CP gene constructs have been

developed by the Asgrow Seed Company. One of these transgenic lines (CZW-30) was evaluated

under field conditions at Cornell University (Fuchs et al., 1997). Cantaloupe line CZW-30

expressing the CP genes of CMV, ZYMV, and WMV 2 was tested against infections by CMV,

ZYMV, and WMV 2. In all trials, transgenic plants showed excellent resistance against single or

mixed infections. Transgenic homozygous plants developed localized mild symptoms late in the

growing season whereas all control plants showed severe systemic symptoms and had high virus

titers 5-6 weeks after transplanting. Interestingly, transgenic hemizygous plants also exhibited a

significant delay (2-3 weeks) in the onset of disease compared to control plants. Strikingly, 3-

10% of the homozygous and 31-35% of the hemizygous plants had dual or triple infections

compared to 66-99% control plants which had mixed infections.

High levels of resistance to a wide range of CMV strains was expected in cantaloupe

plants expressing the CMV-WL CP gene based on earlier studies on tobacco (Namba et al.,

1992) and tomato (Provvidenti and Gonsalves, 1995) plants containing this CP gene. However,

transgenic cantaloupe lines expressing the CMV-WL CP gene were not highly resistant to

infection by CMV in the greenhouse (Gonsalves et al., 1994). One of these lines designated H7-

Review of literature

26

21 was further evaluated under field conditions. It showed a 4-wk delay in infection relative to

the control plants, but ELISA performed 9 weeks after planting revealed that 88% of the

transgenic plants accumulated CMV similar to non-transformed controls (98%) (Fuchs et al.,

1997).

Bottle gourd: no information is available on the development of CGMMV resistant bottle

gourd through genetic engineering. Agrobacterium mediated transformation in Cucurbitaceae has

been reported by several workers (Fang and Grumet, 1990; Chee, 1990). Han, et al. (2005)

described procedures for Agrobacterium mediated transformation of bottle gourd by inoculating

cotyledonary explants with glufosinate ammonium-resistance gene and -D-glucuronidase

reporter gene. The most effective bacterial infection was observed when cotyledon explants of

four-day-old seedlings were co-cultivated with Agrobacterium for six to eight days on co-

cultivation medium supplemented with 0.1 to 0.001 mg/ml L- (2-aminoethoxyvinyl) glycine.

The transgenic bottle gourd plants were obtained at frequency of 1.9%.

Watermelon: Genetic engineering approach is used targets improve watermelon

resistance to environmental biotic and abiotic stresses and improving the nutritional quality.

The use of genetic transformation for production of transgenic plants and somaclonal

variation to produce polyploidy plants appear to be the most common biotechnological

approaches for cultivar improvement (Compton et al., 2004). Polyploid watermelons were

found to be resistant to watermelon fruit blotch and nematodes (Garret et al., 1995). Moreover,

in triploid watermelons, orange flesh turns into deeper orange color as it ripens and the

flavour can even improve after harvesting. Their tough sunburn-resistant rind also makes

them excellent for long-distance shipping (Raza et al., 2003). Efficient methods for

regenerating watermelon in vitro have been in place for over a decade (Table 2.6) and

have paved the way for genetic engineering studies through Agrobacterium-mediated and

biolistics-mediated transformation (Compton et al., 2004).

Stable genetic transformation of Citrullus species by Agrobacterium

tumefaciens strain LBA 4404 has been reported using cotyledon explants (Choi et al.,

1994). Recently, Park et al. (2005) have described the successful transformation of a

watermelon rootstock (gongdae) by Agrobacterium tumefaciens-mediated transformation

Review of literature

27

Table 2.6: Comparison of watermelon regeneration and transformation protocols.

Explants Callusing

medium

Shoot

regeneration

medium

Cultivar Used For References

Cotyledon

and leaf

0.5 or 1.0

mg/l 2, 4-D

0.5 or 1.0

mg/l BA

Gizal Reg. (70-75%)

and GUS trans.

(96%)

Ibrahim et

al., 2009

Cotyledon 3.2 mg dm-3

BA, 0.5

mg

dm-3

IBA

0.1

mg dm-3

IBA, 0.5 mg

dm-3

GA3

Daesan GUS-trans.

(1.6%)

Cho et al.,

2008

Cotyledon 4 mg/l BA BA 4mg/l No 101117-

1

GUS Akashi et

al., 2005

Cotyledon 1.0 mg/l BA,

0.2 mg/l IAA

1.0 mg/l BA

+ 0.1 mg/l

IAA

Root stock

gongdae

CGMMV-CP

(0.1-0.3%)

Park et al.,

2005

leaf 2.5 mg/l 2, 4

D

1.0 mg/l BA,

0.2 mg/l

NAA

Thumb Reg. (70%) Sultana et

al., 2004

Cotyledon 5 M BA 20 M BA Crimson Reg-75-78%,

GUS trans.

Yalcin -

Mendi et

al., 2003

Cotyledon 1.0 mg/l BA 1.0 mg/l BA,

0.2 mg/l

kinetin

D. Maravill,

Crimson

sweet, Sugar

baby

Salt tolerance

2.8-5.3%

Ellul et al.,

2003

Cotyledon 3.0 M BA,

3.0 M 2iP

(dir.shooting)

3.0 M BA,

3.0 M IAA

(Callusing)

1.0 M BA Sugar baby Reg. 55% Chaturvedi

and

Bhatnagar

2001

Reg: regeneration, Trans: Transformation

Review of literature

28

with the CGMMV-CP gene for developing virus resistance watermelon plant. This was the

first report on the production of transgenic watermelon with virus resistance.

A transformation of wild watermelon plants has also been reported by Akashi et al.

(2005). The g r o u p indicated that the use of kanamycin as a selectable marker resulted in

higher number of transgenic shoots produced as compared to selection using hygromycin.

Recently, a method to transform Citrullus lanatus Thumb (cv. Daesan) using Agrobacterium

tumefaciens was reported by Cho et al. (2008) and Ibrahim et al. (2009).

Transformation efficiency of plants using Agrobacterium-mediated is influenced by

several factors such as addition of phenolic compounds (e.g., a cetosyringone) in the co-

cultivation medium and wounding treatment of the target tissue with sonication, glass beads or

biolistics gold particles. Different Agrobacterium strains have also been evaluated to

produce the best transformation system for Citrullus lanatus (Cho et al., 2008). To date, most of

the published methods for transformation of watermelon via Agrobacterium-mediated use

cotyledon sections as the target tissues or explants (Choi et al., 1994; Ellul et al., 2003;

Akashi et al., 2005; Park et al., 2005).

Genetic transformation of watermelon (Citrullus lantalus) cultivar Sugar

baby is a potentially impor tan t tool to improve traits such as disease and herbicide

resistance without altering the cultivar identity. Recently, a method to regenerate shoots from

Sugar Baby Citrullus lantullus cultivars has been reported by Rakhi Chaturvedi et al, (2001).

An effect method for regeneration has been designed after comparative study of previous reports

(Table 2.6)

Review of literature

29

Fig 2.1: Global watermelon production growth (Source: FAOSTAT).

Review of literature

30

Fig.2.2: Worldwide distribution of tospoviruses.

Virus acronyms: CaCV, Capsicum chlorosis virus; CCSV, Calla lily chlorotic spot virus;CSNV,

Chrysanthemum stem necrosis virus; GRSV, Groundnut ringspot virus; GBNV,Groundnut bud

necrosis virus; INSV, Impatiens necrotic spot virus; IYSV, Iris yellow spot virus;MSMV, Melon

severe mosaic virus; MYSV,Melon yellowspot virus; PCFV, Peanut chlorotic fanspot virus;

PoRSV, Polygonum ringspot virus; PSMV, Physalis silver mottle virus; PYSV, Peanut yellow

spot virus; TCSV, Tomato chlorotic spot virus;TSWV, Tomato spotted wilt virus; TYFRV,

Tomato yellow fruit ring virus; TZSV, Tomato zonate spot virus; WBNV,Watermelon bud

necrosis virus;WSMV,Watermelon silver mottle virus; ZLCV, Zucchini lethal chlorosis virus.

Two potentially new, yet to be characterized tospoviruses from Australia. TFYRV, Tomato fruit

yellow ring virus, is considered as an isolate of Tomato yellow ring virus (TYRV).

Review of literature

31

Fig. 2.3: Distribution pattern of Watermelon bud necrosis virus in different states of India.

Review of literature

32

Fig.2.4: Life cycle of Tospoviruses.

Review of literature

33

Fig.2.5.Diagram of TSWV virion.

A double-layered membrane of host origin (blue) is shown with the viral-encoded proteins GN

and GC (green) projecting from the surface in monomeric and dimeric configurations. The

genomic RNA is presented as non-covalently closed circles in the form of a ribonucleoprotein

(RNP) complex created by its association with many copies of N protein (peach). A few copies

of the virion-associated RNA-dependent RNA polymerase (RdRp or L) are shown (purple) in

association with the RNPs (Whitfield, 2005).

Review of literature

34

Fig. 2.6: Schematic transcription and translation by different segment of genomic RNAs of

the Tospovirus.