6

CHAPTER-2 REVIEW OF LITERATURE

2.1 CAPSICUM (BELL PEPPER)

Capsicum (Bell pepper) is one of the important nutritious, highly remunerative

vegetables cultivated in most parts of the world especially in tropical and subtropical regions

of Asian continent, temperate regions of Central and South America and European

countries. Particularly, India, Malaysia, Thailand, Pakistan, Indonesia, Philippines, tropical and

north Africa and South America are the major capsicum producing countries of the world (Tindal,

1983). It is one of the important vegetable crops of family Solanaceae after tomato and potato.

Capsicum (including hot pepper) grown worldwide in an area of 17, 03, 486 hectares with a

production of 2, 60, 56,900 tonnes (FAO, 2009). China is the largest capsicum producing country

in the world. India stands fourth on world production of capsicum with an average annual

production of 0.9 million tons from an area of 0.885 million hectare with a productivity of 1266

kg per hectare (Sreedhara et al., 2013). In Himachal Pradesh, bell pepper has an important place

among the commercial vegetable crops. As far as remuneration is concerned it ranks third after

pea and tomato (Dar et al., 2013). Capsicum may comprise up to 30 species among them five (C.

annuum L., C. frutescens L., C. chinese Jacq., C. baccatum L., and C. pubescens Ruiz and Pavon)

are domesticated species (Wang and Bosland, 2006). Out of these five species, Capsicum annuum

is most cultivated species. It includes both sweet and hot pepper fruits. It is grown mainly for its

green fruits but now day’s different colour hybrids (red, yellow and orange) are also available.

They require mild climate for growth and development. They are different from hot peppers

commonly known as chillies. It is difficult to obtain higher yields of good quality fruits

throughout the year under open conditions in most parts of India. The cultivation of bell

pepper is possible even during the off season under greenhouse conditions. These protected

structures are important for improving their cultivation. Protected structures acts as a physical

barrier and play a key role in Integrated Pest Management (IPM) by preventing spreading of

insects, pests and viruses causing severe damage to crops (Singh et al., 2003). Pepper has both

medicinal and nutritional value as it consists of various vitamins (A, C, B1 and B2) and

minerals such as iron, calcium, magnesium, sulphur and phosphorous (Berke, 2002). Pepper

has also been used as therapeutic agent for cancer (Hartwell, 1971). It has antibacterial

property which helps in indigestion, abdominal pain, constipation and arthritis and also known

7

to stimulate the taste buds which signal the body to produce hydrochloric acid that helps to

breakdown the food not being digested (Pipion et al., 1999; Achiangia et al., 2013). In

addition to this, pepper has also been used in pharmaceutical industries (Achiangia et al.,

2013).

2.2 VIRUSES INFECTING BELL PEPPER

Sweet peppers are susceptible to a number of pathogens which includes bacteria, fungi and

viruses, causing considerable economic looses. According to a survey conducted by Asian

Vegetable Research and Development Center (AVRDC) in 1987 revealed that pepper production in

the tropics is constrained by a number of factors. Viral diseases are considerably important and

gained attention because they are difficult to control and they impose significant production

constraints affecting both yield and quality (Nono-Womdim, 2001). Viruses are intracellular

pathogenic particles that infect living organisms. Viral diseases constitute the major limiting factor

in pepper cultivation throughout the world (Florini and Zitter, 1987; Martelli and Quacquarelli,

1983; Green and Kim, 1991). About 68 viruses have been reported to infect pepper from various

part of the world in terms of frequency of distribution, damage and host range (Pernezny et al.,

2003). In India, 19 different viruses have been reported to cause natural infection. Among them 13

viruses have been characterized and classified as potyviruses, potexviruses, cucumoviruses,

tobamoviruses, tospoviruses, nepoviruses carlaviruses and geminiviruses. Mechanically transmitted

viruses like tobamoviruses are predominant in protected crops. Aphid transmitted viruses include

Potato virus Y (PVY) , Pepper veinal mottle virus (PVMV), Tobacco etch virus, (TEV), Pepper

vein banding virus (PVBV), Chili veinal mottle virus (CVMV), Pepper mottle virus (PMV), Pepper

severe mosaic virus (PeSMV), Pepper yellow mosaic virus (PYMV), Chilli vein banding mottle

virus (CVbMV) and Cucumber mosaic virus (CMV) belongs to family Potyviridae and

Bromoviridae respectively. Nematode transmitted viruses include family Comoviridae and virus are

Tobacco ring spot virus (TRSV), Tomato ring spot virus (TomRV) and Tomato black ring virus

(TBRV) and whitefly transmitted viruses from family Geminiviridae including Tobacco leaf curl

virus (TLCV), Curly top virus (CTV), Tomato yellow leaf curl virus (TYLCV) , Tomato chlorosis

virus (TChV), Pepper mild tigre virus (PMTV), Pepper hausteco virus (PHV), Serrano golden

mosaic virus (SGMV), Tomato dwarf leaf curl virus (ToDLCV), Chino del tomate virus (CdTV).

Tobamoviruses include Tobacco mosaic virus (TMV), Pepper mild mottle virus (PeMdMtV),

Tomato mosaic virus (ToMV), Bell pepper mottle virus (BPeMtV), Paprika mild mottle virus

8

(PMMV), tobraviruses include Tobacco rattle virus (TRV), and Pepper ring spot virus (PRSV).

Tospoviruses include Tomato spotted wilt virus (TSWV), Groundnut ring spot virus (GRSV),

Impatiens necrotic spot virus (INSV), Groundnut bud necrosis virus (GBNV) and Capsicum

chlorosis virus (CaCV).

2.3 TOSPOVIRUSES

Tospoviruses are enveloped viruses belonging to family Bunyaviridae. Tospovirus is

the only plant infecting genus of this family; other viruses in this family exclusively infect

animals. Brittlebank, (1919) published the first description of this new disease, detected on

tomatoes in 1915 in the state of Victoria (Australia) and he called it “spotted wilt of tomato”. In

the same year, Osborn also observed this disease on tomatoes in the south of Australia and in

1920 its presence was reported in all the Australian states (Best, 1968). The first characterization

of this virus as the causal agent of the disease was reported by Samuel et al. (1930), who gave it

its current name “Tomato spotted wilt virus”. Since then it was reported in several tropical and

temperate regions and it is considered worldwide in distribution. TSWV ranks second in the top

ten most detrimental viruses worldwide (Scholthof et al., 2011). Tospoviruses are spherical

particles with 80-120 nm diameter range and unique among plant viruses in that virions are

enveloped in a host derived membranes with two glycoproteins. These glycoproteins (GPs) and

are the major determinants of specificity and transmission by the thrips vectors (Sin et al., 2005;

Ullman et al., 2005). All members of Tospovirus genus are distinguished on the basis of N

protein serology, N protein RNA sequence and vector specificity for their movement in

respective hosts (Goldbach and Kuo, 1996). There was a report showing that more than 20

(accepted and tentative) tospoviruses have been reported from all over the world (Pappu et al.,

2009). The species included in genus Tospovirus currently includes, Tomato spotted wilt virus

(TSWV), Tomato chlorotic spot virus (TCSV), Groundnut ring spot virus (GRSV), Impatiens

necrotic spot virus (INSV), Groundnut bud necrosis virus (GBNV), Watermelon silver mottle virus

(WSMoV), Peanut yellow spot virus (PYSV), Zucchini lethal chlorosis virus (ZLCV),

Chrysanthemum stem necrosis virus (CSNV), Iris yellow spot virus (IYSV), Peanut chlorotic

fan-spot virus (PCFV), Melon yellow spot virus (MYSV), Watermelon bud necrosis virus

(WBNV), Tomato yellow fruit ring virus (TYFRV) {synonym: Tomato yellow ring virus

(TYRV)}, Calla lily chlorotic spot virus (CCSV), Capsicum chlorosis virus (CaCV), Alstroemeria

necrotic streak virus (ANSV), Bean necrotic mosaic virus (BeNMV), Groundnut chlorotic fan-

spot virus (GCFSV) and Zucchini lethal chlorosis virus (ZLCV). Three new putative species,

9

Soybean vein necrosis-associated virus (Zhou et al., 2011), Bean necrotic mosaic virus (de

Oliveira et al., 2011) and Pepper necrotic spot virus (Torres et al., 2012), have recently been

described. Even in the year 2014, two new tospoviruses Pepper chlorotic spot virus (PCSV)

and Lisianthus necrotic ringspot virus (LNSV) have also been reported (Cheng et al., 2014;

Shimomoto et al., 2014).

2.4 TOSPOVIRUSES REPORTED FROM INDIA AND THEIR INTERNATIONAL

STATUS

Based on the nucleocapsid (N) protein characteristics, currently, five tospoviruses have

been reported from India viz. GBNV/PBNV, CaCV, IYSV, PYSV and WBNV (Table 1).

Groundnut bud necrosis virus (GBNV) (Reddy et al., 1992; Satyanarayana et al., 1996) from

groundnut/peanut (Arachis hypogaea) and many other plants, such as tomato (Solanum

lycopersicum), pea (Pisum sativum), cowpea (Vigna unguiculata), mungbean (Vigna radiata),

soyabean (Glycine max) and potato (Solanum tuberosum) (Bhatt et al., 2002; Umamaheswaran et

al., 2003; Thien-Xuan et al., 2003; Jain et al., 2004; Akram et al., 2004; Raja and Jain, 2006;

Akram et al., 2010), Watermelon bud necrosis virus (WBNV) from watermelon (Jain et al.,

1998), Groundnut yellow spot virus (GYSV) (Satyanarayana et al., 1998) from groundnut,

Irish Yellow spot virus (IYSV) (Ravi et al., 2006) from onion and Capsicum chlorosis virus

(CaCV) from tomato and chilli pepper (Kunkalikar et al., 2007; Krishnareddy et al., 2008;

Kunkalikar et al., 2010).

Table: 2.1 Tospoviruses reported from India (Taken and modified from Mandal et al., 2012)

Virus Crop

Infected

Place of

emergence

Key

symptoms

References

Groundnut bud necrosis

virus (GBNV)

Groundnut Andhra Pradesh Bud necrosis Reddy et al.,

1992

Peanut yellow spot virus

(PYSV)

Groundnut Andhra Pradesh Yellow spot Satyanaryana et

al., 1998

Watermelon bud necrosis

virus (WBNV)

Watermelon Karnataka Bud necrosis Jain et al., 1998

Iris yellow spot virus

(IYSV)

Onion Andhra Pradesh Chlorotic

lesions

Ravi et al.,

2006

Capsicum chlorosis virus

(CaCV)

Chilli Karnataka Apical

necrosis

Krishnareddy et

al., 2008

10

2.4.1 Groundnut bud necrosis virus (GBNV)

Groundnut bud necrosis virus (GBNV) also known as Peanut bud necrosis virus

(PBNV) as its disease was first described in peanut in 1968 (Reddy et al., 1968; 1992). It is

the most ubiquitous virus among the five tospoviruses reported from the Indian subcontinent

(Mandal et al., 2012). In recent years, GBNV has been reported to infect crops such as

soybean (Bhat et al., 2002), mung bean (Thien-Xuan et al., 2003), tomato (Umamaheswaran et

al., 2003) and potato (Jain et al., 2004). The estimated annual losses caused by GBNV were

reported to be over US $89 million (Reddy et al., 1995; Mandal et al., 2012). 70-90% losses were

reported for peanut (Singh and Srivastava, 1995) and 29% for potato (Singh et al., 1997) have been

reported (Akram et al., 2012). Similarly, 100% GBNV disease incidence was found in tomato in

Maharashtra, Andhra Pradesh and Karnataka states of India (Kunkalikar et al., 2011). GBNV

found to infect various plant species belonging to families such as Chenopodiaceae,

Amaranthaceae, Asteraceae, Cucurbitaceae, Fabaceae, Malvaceae, and Solanaceae (Thien-Xuan et

al., 2003; Raja, 2005; Saritha, 2007; Mandal et al., 2012). In Thailand, GBNV was also found in

peanut (Wongkaew, 1995; Chiemsombat et al., 2008).

2.4.2 Watermelon bud necrosis virus (WBNV)

Watermelon bud necrosis virus (WBNV), infecting watermelon (Citrullus lanatus) was

first reported from southern India (Singh and Krisnhareddy, 1996). From states, Karnataka,

Andhra Pradesh and Maharashtra, 39-100% disease incidence was reported for WBNV with an

estimated 60-100% yield loss (Krisnhareddy and Singh, 1993). In addition to watermelon,

WBNV have been reported to infect Luffa acutangula (ridge gourd) (Mandal et al., 2003),

three cucurbits and three fabaceous crops (cowpea, frenchbeans and sem) (Jain et al., 2007)

with an estimated yield losses up to 100%. Recently, thus virus has also been found in chilli

and tomato in northern India (Kunkalikar et al., 2011). Based on the host range, symptoms and

serological testing, the casual agent was identified as a new tospovirus and named as WBNV

(Singh and Krishnareddy, 1996). GBNV and WBNV are the most economically important

viruses reported affecting crops like peanut, tomato, potato and cucurbitaceous crops (Singh and

Srivastava, 1995; Reddy et al., 1995; Singh et al., 1997; Jain et al., 2007). Based on the NP gene,

GBNV and WBNV are currently assigned to the Watermelon silver mottle virus (WSMoV)

serogroup (Fauquet et al., 2005).

11

2.4.3 Peanut yellow spot virus (PYSV)

Peanut yellow spot virus (PYSV) was first reported in peanut and was

described as a disease that causes necrosis due to chlorotic/yellow leaf spots infecting 90% of the

fields (Mandal et al., 2012). It was also known by synonym Groundnut yellow spot virus

(GYSV). The disease was again reported in 1991 as a distinct tospovirus causing systemic

infections in peas, mung bean and cow pea (Reddy et al., 1991), and subsequently the disease

was serologically tested and confirmed as new species on the basis of S RNA sequence. GYSV

(Satyanarayana et al., 1996, 1998) was found to be serologically different from TSWV, INSV and

GBNV.

2.4.4 Iris yellow spot virus (IYSV)

Iris yellow spot virus (IYSV) has been reported in onions in Jalna and Nasik regions of

Maharashtra, the major onion producing state in the country in 2006 (Ravi et al., 2006) but no

economic losses have been reported. IYSV is a recently emerging tospovirus, with outbreaks

in onions recorded from Spain (Cordoba-Selles et al., 2005), Germany (Leinhos et al., 2007),

Serbia (Bulajic et al., 2008), Greece (Chatzivassiliou et al., 2009), Italy (Tomassoli et al.,

2009), UK (Mumford et al., 2008), and Netherlands (Hoedjes et al., 2011).

2.4.5 Capsicum chlorosis virus (CaCV)

Recently, Capsicum chlorosis virus (CaCV) has been reported infecting tomato

(Kunkalikar et al., 2007) and chili pepper (Krishnareddy et al., 2008). Capsicum chlorosis virus

(CaCV) was first reported from Australia in Capsicum spp. (MacMichael et al., 2002). In India, it

was reported from southern, central and northern parts. Among these, it was first reported in 2007

from tomato in northern India (Kunkalikar et al., 2007) and chilli pepper in Bangalore (Karnataka

State) (Krishnareddy et al., 2008). Similar infection of CaCV has been reported from Thailand in

the crop members of Solanaceae family (Knierim et al., 2006; Chiemsobat et al., 2008) and in

tomato (Premachandra et al., 2005). In Taiwan, CaCV was also reported from Phalaenopsis

orchids (Zheng et al., 2008), on tomato (Huang et al., 2010), Calla lilies (Zantedeschia spp.) (Chen

et al., 2007a), Blood lily (Haemanthus multiflorus) and amaryllis (Hippeastrum hybridum) (Chen

et al., 2009). From Taiwan, it has also been reported in sweet pepper (Zheng et al., 2010). It has

also been reported from peanut (Arachis hypogaea L.) in China (Chen et al., 2007b) and in

12

Thailand (Chiemsobat et al., 2008). CaCV has also been reported from capsicum from Kununurra,

WA in 2004 (Jones and Sharman, 2005). In USA, CaCV has also been reported from Wax flower

(Hoya calycina Schlecter) (Melzer et al., 2014). Peanut, Hoys and several weed species have been

reported as natural hosts of CaCV. CaCV has also been reported from Amaranthus one of its

natural host from Himachal Pradesh, India (Sharma and Kulshrestha, 2014). CaCV is a member of

the Watermelon silver mottle virus or sergroup IV tospoviruses which are prevalent in Asia

(McMichael et al., 2002).

2.5 HOST RANGE AND SYMPTOMATOLOGY OF TOSPOVIRUSES

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

Van et al., 1993; Gognalons et al., 1996; Verhoeven and Roenhort, 1998; Chatzivassiliou et al.,

1999). TSWV is present throughout the world and infects a wide range of plants, with more

than 1300 plant species - dicots and monocots, crop plants, ornamentals and weeds-susceptible

to this virus (Peters, 2003). Most of the plant species susceptible to TSWV belong to the families

Asteraceae and Solanaceae. INSV also has a broad host range of more than 300 species including

dicots and monocots (Pappu et al., 2009). Although INSV presents a serious problem to the

ornamentals industry (Daughtrey et al., 1997; Elliott et al., 2009), the virus can occasionally also

infect, at a low level, field crops such as lettuce, cucumber, pepper (Vicchi et al., 1999) and potato

(Perry et al., 2005).

GBNV can infect more than 800 plant species, in more than 80 families including both

dicots and monocots. It has been reported that GBNV infects the members of families

Amaranthaceae, Asteraceae, Chenopodiaceae, Cucurbitaceae, Compositae, Fabaceae,

Solanaceae and Malvaceae (Thien- Xuan et al., 2003; Raja, 2005; Saritha, 2007 ; Mandal et al.,

2012). Compositae and Solanaceae have largest range of host plants susceptible to GBNV (Prins et

al., 1996). WBNV was moderately related to Indian GBNV on serology basis but host range is

very much different 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). Generally, CaCV have limited natural host range (Persley et al., 2006; Lebas and Ochoa-

Corona, 2007) similar as that of GBNV (Mandal et al., 2012). The crop hosts of CaCV are

capsicum (including chilli types), tomato and peanut. The virus has also been found in the weed

13

species Ageratum conyzoides (Billygoat weed), Sonchus oleraceus and Emilia sonchifolia (Emilia

or purple sowthistle). Virus incidence in Ageratum is often high, indicating that this weed may play

an important role in the survival and dispersal of CaCV. Although the symptoms of CaCV

resemble those caused by TSWV, there are several distinct features. In capsicum, chlorosis or

yellowing on leaf margins and between the veins develops on young leaves, which often become

narrow and curled, with a strap-like appearance. Older leaves become chlorotic with ring spots and

line patterns. The fruit on infected plants are small, distorted and often marked with dark spots and

scarring over the surface. Infected tomato plants develop chlorotic spots and blotches on leaves

which become mottled and stunted with death of some leaves. Fruit from infected plants develop

chlorotic rings and necrotic areas. Symptoms in ornamentals vary significantly since local and

systemic infections depend on the host species. On some hosts, symptoms can be found on few

leaves only (Baker et al., 2007; Zheng et al., 2008), e.g. chrysanthemum, while on other hosts with

systemic infection, spots and rings on leaves and systemic necrosis are observed (Kritzman et al.,

2000). On leaves, the most striking symptoms indicating tospovirus infection are concentric

chlorotic to necrotic rings or ring patterns, which can also be found on stems (Daughtrey et al.,

1997). On stalks and bulbs of Allium spp. necrotic and/or chlorotic lesions (diamond shape),

twisting and bending of flower-bearing stalks mark infections with IYSV (Persley et al., 2006).

Although genetically distinct, most of these viruses cause symptoms similar to those

associated with TSWV infection, with stunted plants, chlorotic and necrotic spots on leaves

and petioles and a range of symptoms on fruits leading to unmarketable products. Although

quantitative data on yield loss in crops and ornamentals are generally missing for these

viruses, for tomato at least losses similar to those associated with TSWV diseases can be

assumed. Moreover, serious consequences resulting from infections with tospoviruses other

than TSWV in tomato and pepper can arise from breaking introgressed resistance, as

reported for TSWV resistance Sw-5 (Jahn et al., 2000). In young plants, WBNV has been shown to

cause rapid dieback, and wilting of plants develop dramatically causing a total loss in the affected

plants and in mature crop, shortened internodes, upright growth of younger shoots, necrosis on

stem, petiole, and fruit stalk are commonly seen (Mandal et al., 2012). In GBNV, mild chlorotic

spots appear on young leaves, and subsequently necrosis and chlorotic rings develop (Reddy et al.,

1991). GBNV was reported as the most economically important virus affecting a variety of crops

such as peanut, potato, tomato, soybean, urdbean, mungbean and cowpea (Akram et al., 2004; Jain

et al., 2007; Pappu et al., 2009).

14

2.6 GENOME ORGANIZATION AND FUNCTIONS OF DIFFERENT GENES

Tospoviruses form a unique genus in the family Bunyaviridae, having a tripartite genome

of ssRNA molecules comprising a total of five open reading frames (ORFs) (Knierim et al.,

2006). The negative polarity L RNA is 8897 nucleotides long whereas ambisense M and S RNAs

are approximately 4821 and 2916 nucleotides long respectively (de Hann et al.,1990; 1991;

Kormelink et al., 1992a). The complete nucleotide sequences were reported for INSV (de Haan

et al., 1992, Law et al., 1992; Van Poelwijk et al., 1997), GBNV (Satyanarayana et al., 1996a;

Satyanarayana et al., 1996; Gowda et al., 1998), WSMoV (Yeh et al., 1996; Chu and Yeh, 1998;

Chu et al., 2001), MYSV (Kato et al., 2000; Okuda et al., 2006) and CaCV (Knierim et al.,

2006).

2.6.1 L RNA

The largest RNA (L RNA) is negative sense and monocistronic. L RNA serves as a

multifunctional, replication-associated protein and is believed to function cooperatively with

host-encoded factors. The largest RNA (L RNA) was found to be in the range of 8.9 to 9 kb for

TSWV (de Haan et al., 1991), GBNV (Gowda et al., 1998), WBNV (Li et al., 2010) and CaCV

(Knierim et al., 2006; Kunkalikar et al., 2010). It has a single open reading frame (ORF) in the

viral complementary sense. The RNA is 8897 nucleotides long coding for a 330 kDa protein,

which is the putative RNA-dependent RNA polymerase (RdRp) or L protein (de Haan et al.,

1991; Lee et al., 2011). This 330-kDa protein has been implied in several enzymatic activities

such as transcriptase, replicase and endonuclease (Adkins et al., 1995; Van Poelwijk, 1996;

Chapman et al., 2003). RNA viruses show extremely high mutation rates, because of lack of

proofreading ability of their polymerases (Moya et al., 2000). Although, the mutation or error

rate of viral RNA-dependent RNA polymerase (RdRp) has not been estimated for plant viruses,

it has been measured for animal RNA viruses and it is approximately 10–4

, or one error per

genome per replication cycle (Roossinck, 1997). As tospovirus has both negative and ambisense

coding strategies, the RNA dependent RNA polymerase has to be co transported with the viral

RNA to allow transcription and replication within the newly infected cells (Soellick et al., 2000).

2.6.2 M RNA

The M RNA is approximately 4.8 kb (Lee et al., 2011) for TSWV, GBNV (Groundnut

isolate) and CaCV (Satyanaryana et al., 1996; Kunkalikar et al., 2010), 4.7 kb for WBNV

15

(Kumar et al., 2010; Li et al., 2010) and encodes a 34 kDa protein (TSWV) and 34.3 kDa

(GBNV) in viral sense designated nonstructural NSm (commonly called as movement protein)

proposed to be involved in cell to cell movement of nonenveloped ribonucleocapsid structures

(Storms et al., 1995; Silva et al., 2001; Soellick et al., 2000) and stimulation of tubule formation

in protoplasts. Plant viruses have to overcome the barrier of cellulose containing cell walls to

establish a successful systemic infection on the plant host. So, viral movement proteins evolved

that facilitate transport of infectious material through plasmodesmata, the intercellular

connection for the plant cell (Griffiths et al., 1992). In plant tissue the NSm protein of TSWV

functions as viral movement protein (MP), aggregating into plasmodesmata-penetrating tubules

to establish cell-to-cell movement. As upon heterologous expression NSm was able to form

similar tubules on the surface of insect (Spodoptera frugiperda) cells, expression and cellular

manifestation of this protein in infected thrips tissue was also investigated. It was shown that

NSm, though detectably expressed in both the L2 larval and adult thrips stages, does not

aggregate into tubules, indicating that this requirement was associated to its function as MP in

plants, and raising the question if NSm has a function at all during the insect life cycle of TSWV

(Storms et al., 2002).

TSWV NSm domains required for tubule formation, movement and symptoms were

identified previously by deletion-mapping and alanine-substitution mutagenesis using the TMV-

based system. Mutagenesis studies of TSWV NSm amino acids that are conserved in other

tospovirus were conducted by Li et al. (2010) and suggested that functional domains of NSm

protein may be conserved across the genus. Recent findings suggest that these movement

proteins, which recognize and transport the viral genomes as naked nucleic acid or in complex

with other viral proteins, resemble plant proteins that are involved in selective trafficking of

protein and protein- nucleic acid complexes through plasmodesmata as part of fundamental

transport and signaling process (de Haan et al., 1990). The molecular basis of NSm function was

studied by expressing the protein in Escherichia coli and investigated protein-protein and

protein-RNA interactions of NSm protein in vitro. NSm specifically interacts with TSWV N

protein and binds single-stranded RNA in a sequence-nonspecific manner. Using NSm as bait in

a yeast two-hybrid screen, two homologous NSm-binding proteins of the DnaJ family from

Nicotiana tabacum and Arabidopsis thaliana were identified (Soellick et al., 2000). The viral

complementary sense (vc RNA) ORF codes for glycoprotein precursor GP, which is post-

16

translationally cleaved into the spikes or glycoproteins G1 and G2 (Kormelink et al., 1992a). For

TSWV, the 58 kDa G2 is also referred to as G(N) and 78 kDa G1 as G(C) (N and C refer to the

amino and carboxy terminal position within the glycoprotein precursor, respectively) (Snippe et

al., 2007). These glycoproteins are required for virus infection of the arthropod vector. Other

members of the Bunyaviridae enter host cells by pH-dependent endocytosis. During this process,

the glycoproteins are exposed to conditions of acidic pH within endocytic vesicles causing the

G(C) protein to change its conformation. This conformational change renders G(C) more

sensitive to protease cleavage. TSWV virions were subjected to varying pH conditions and

determined that TSWV G(C), but not G(N), was cleaved under acidic pH conditions and this

phenomenon was not observed at neutral or alkaline pH. This provides evidence at low pH, G(C)

conformation changes, which results in altered protease sensitivity. Furthermore, sequence

analysis of G(C) predicts the presence of internal hydrophobic domains, regions that are

characteristic of fusion proteins (Whitfield et al., 2005).

The presence of the membrane glycoproteins was found to be essential for the virus’s

ability to replicate alternately in its plant host and its thrips vector (Wijkamp, 1996). Evidence

for the involvement of the glycoproteins in thrips transmission was provided by the interaction of

the glycoproteins with the proteins of the thrips vector (Bandla et al., 1998), the loss of thrips

transmissibility of envelope-deficient mutants (Resende et al., 1991) and the presence of a

sequence motif that is characteristic for cellular attachment domains (Kormelink et al., 1992a).

TSWV glycoproteins were also reported to induce the formation of endoplasmic reticulum and

Golgi-derived pleomorphic membrane structures in plant cells (Ribeiro et al., 2008). Interactions

between TSWV glycoprotein’s and nucleocapsid (N) proteins were studied using Fluorescence

resonance energy transfer (FRET) and Fluorescence lifetime imaging microscopy (FLIM)

techniques. Interaction was demonstrated between G (C) and N and not in G (N) and N using

both the techniques (Snippe et al, 2007). Recently by using FLIM technique, it has been studied

that nucleocapsid protein interacts with both viral glycoproteins (Riberio et al., 2009).

Glycoproteins G (N) and G (C) were examined for their lectin binding affinity (Mannose binding

lectins, N-Acetyllactoseamine lectins and fucose binding lectins) and their sensitivities to

glycosidases to know the nature of present oligosaccharides residues on them. Result showed

that G (C) showed strong binding to three lectin molecules whereas G (N) has lesser affinity to

mannose lectin but not to other two.

17

Fig. 2.1: Replication strategy for tripartite genome of tospoviruses (data taken from de

Haan et al. 1990, 1991; de Aliva 1992; Kormelink et al., 1992c; Roselló et al.,

1996).

18

After treatment with two glycosidases (endoglycosidase H and peptide:N-glycosidase F

(PNGase F), there was significant decrease in the binding of G (C) but no such effect was

observed on G(N). Due to difference in binding properties of two glycoproteins it has been

suggested that glycoprotein G (C) is heavily glycosylated as compare to G (N). There is no

evidence observed for the presence of O-linked oligosaccharides on G (N) or G (C) (Naidu et al.,

2004). During viral infection of a plant cell, the two glycoproteins eventually accumulate in the

Golgi complex. So, site of TSWV particle morphogenesis was determined to be the Golgi system

of host plant (Kikkert et al., 1999). Golgi stacks containing the two glycoproteins then wrap

around viral ribonucleoprotein (RNP) complexes consisting of viral RNA, associated with

nucleocapsid (N) protein and the putative viral RNA-dependent RNA polymerase (RdRp) to

form doubly enveloped virus particles. These are thought to fuse with each other and with ER-

derived membranes, resulting in the formation of large intracellular vesicles containing singly

enveloped virus particles (Kikkert et al., 1999). The formation of the enveloped virus particles is

strongly regulated by the viral glycoproteins. They generally accumulate independently at a

particular cellular membrane by targeted transport through the secretary pathway, to facilitate the

interaction with the viral nucleocapsids and the initiation of budding (Stephens and Compans,

1988; Petterson 1991; Griffiths et al., 1992).

2.6.3 S RNA

The S segment was found to be approximately 3 kb and contains two ORFs in ambisense

orientation separated by a large intergenic region. It was reported to be 3.05 kb for GBNV

(Satyanaryana et al., 1996), 3.4 kb for WBNV (Li et al., 2010), 3.1 kb for CaCV (Kunkalikar et

al., 2010). The ORF nearer the 5′ end of the RNA, codes for a nonstructural protein in the viral

sense designated NSs (54 kDa). The role of the NSs protein has long been enigmatic, but

recently NSs was shown to be a suppressor of gene silencing required to protect the virus against

the plant's anti-viral response of post-transcriptional gene silencing (PTGS) and also affects

symptom expression in TSWV-infected plants (Takeda et al., 2002; Bucher et al., 2003). TSWV

NSs was the first RNA silencing suppressor identified in negative-strand RNA viruses. TSWV

NSs protein suppressed sense transgene-induced PTGS but did not suppress inverted repeat

transgene- induced PTGS (Takeda et al., 2002). Recently, biochemical analysis of NSs proteins

from different tospoviruses using purified NSs or NSs containing cell extracts showed that NSs

proteins showed affinity to small double stranded RNA molecules i.e., small interfering RNAs

19

(siRNAs) and micro-RNA (miRNA). The NSs protein of TSWV was shown to be capable of

inhibiting Dicer- mediated cleavage of long dsRNA in vitro. In addition, it suppressed the

accumulation of green fluorescent protein (GFP)-specific siRNAs during co infiltration with an

inverted-repeat-GFP RNA construct in Nicotiana benthamiana (Schnettler et al., 2010). The

ORF near the 3′ end is in viral complementary (vc) sense and encodes for the nucleocapsid

protein (N) (29 kDa) which encapsidates the viral RNAs within the viral envelope (de Haan et

al., 1990). The nucleocapsid protein (N) contributes to the viral replication cycle in a structural

and, perhaps, regulatory manner by participating in the complex interactions among the RNP

components leading to the initiation of viral RNA transcription and replication. Consistent with

its role in fulfilling this putative function, the N protein has been shown to form dimers in the

absence of RNA (Uhrig et al., 1999; Kainz et al., 2004) and to cooperatively bind ssRNA but not

dsRNA (Richmond et al., 1998). So, each RNA segment of tospovirus is associated with

nucleocapsid (N) proteins (29kDa) and a few copies of the large (L) protein to form pseudo-

circular nucleocapsid (N) structures or ribonucleic protein particles (RNPs) (Kormelink et al.,

1992a; Peters, 2003). These particles result from the homopolymerization of the N protein, and

are highly stable in plant cells and can be easily purified from TSWV infected plant cells by

ultracentrifugation (de Aliva et al., 1990). On the basis of these two properties of N protein

(homopolymerization and high stability), a gene fusion approach was explored to increase the

stability of foreign proteins produced in plants, thereby offering a possible purification

alternative for a target protein as a gene fusion. These results show that the homopolymerization

properties of the N protein can be used as a fast and simple way to purify large amounts of

proteins from plants (Lacorte et al., 2007). By using Fluorescence resonance energy transfer

(FRET) and Fluorescence lifetime imaging microscopy (FLIM) techniques homotypic

interactions of nucleocapsid proteins were studied (Snippe et al., 2005). Mutated forms of N

protein serve as potent dominant-negative inhibitors of virus replication (Rudolph et al., 2003).

The complete nucleotide sequence of the TSWV is now available, allowing the precise

comparison with the other animal infecting members of the Bunyaviridae family and with other

families of plant infecting viruses.

2.6.4 Intergenic region (IGR)

The oppositely located ORFs on the ambisense S and M RNA segments are separated by

intergenic regions (IGRs) of several hundred nucleotides and are regarded as the most hyper

20

variable regions of the genome. Analysis of the intergenic region (IGR) of S and M RNAs of

tospoviruses (Family Bunyaviridae) indicated their heterogeneity both in length and sequence. Both

IGRs contain a long stretch of mainly A residues followed by a long stretch of mainly U residues, and

are predicted to form large stable hairpin structures (~120 bp for the S RNA, ~75 bp for the M

RNA;) (de Haan et al., 1990; Kormelink et al., 1992a). In addition, a sequence (CCAAUUUGG

for S and GCAAACUUUGG for M) that is conserved between different tospoviruses is located

near the top of these intergenic hairpins (Maiss et al., 1991; de Haan et al., 1992; Kormelink et al.,

1992a). More specifically the 5′ and 3′ends of the IGRs are conserved, separated by variable

sequences, deletions and insertions that appear as gaps in alignments (Bhatt et al., 1999; Heinze

et al., 2001). From the estimated sizes of the mRNAs, transcription is thought to terminate

somewhere in the IR (de Haan et al., 1990; Kormelink et al., 1992a), and it has been suggested

that the hairpin structure or the conserved sequence motif may be involved in transcription

termination (van Knnipenberg et al., 2005). There was a report on detailed sequence analysis of

intergenic regions (IGRs) of S and M RNAs of known tospoviruses (Pappu et al., 2000). In general,

IGRs of M RNA were shorter in length compared to the IGRs of their respective S RNA species. Per

cent identity among the S RNA IGR sequences of distinct tospovirus species varied from 42 to

57%, whereas it was 79 to 99% among isolates of the same species. Similarly, when IGRs of M

RNAs were compared, there was higher sequence identity among isolates of the same tospovirus

species (84 to 98%) than among distinct tospovirus species (46 to 59%). Per cent nucleotide

identities and maximum likelihood trees of IGR sequences of S and M RNAs indicated that their

sequence divergence was similar to that of nucleocapsid gene at inter and intra-species levels

(Pappu et al., 2000).

The intergenic region (IGR) of the medium (M) RNA of TSWV isolates naturally

infecting peanut (groundnut), pepper, potato, stokesia, tobacco and watermelon in Georgia (GA)

and a peanut isolate from Florida (FL) was analyzed. The IGR sequences were compared with one

another and with respective M RNA IGRs of TSWV isolates from Brazil and Japan and other

tospoviruses. The length of M IGR of GA and FL isolates varied from 271 to 277 nucleotides. IGR

sequences were more conserved (95-100%) among the populations of TSWV from GA and FL,

than when compared with those of TSWV isolates from other countries (83-94%). Cluster

analysis of the IGR sequences showed that all GA and FL isolates are closely clustered and are

distinct from the TSWV isolates from other countries as well as from other tospoviruses (Bhatt et

21

al., 1999). Intergenic regions of CaCV were also reported from Lycopersicum esculentum in

Thailand. It was reported to be 433 nucleotides long for M RNA segment and 1202 nucleotides

long for S RNA segment of CaCV (Knierim et al., 2006).

2.6.5 Transcription

TSWV initiates transcription of their genome by a mechanism called cap snatching. All

TSWV genes are expressed by the synthesis of mRNAs that can be discriminated from the (anti)

genomic RNA strands by the presence of non- viral leader sequences (Kormelink et al., 1992b

and 1992c). For these leader sequences, TSWV has evolved a cap stealing mechanism known as

cap snatching. During this process the viral RNA dependent RNA polymerase (RdRp),

encompassing an endonuclease activity, cleaves a host mRNA at a position 10-20 nucleotides from

the capped 5′ end of host mRNA.

The short capped fragments act as primers for viral mRNA transcription (Kormelink et

al., 1992c; Van Poelwijk et al., 1996; Duijsings et al., 2001). This mechanism was used by all

segmented negative strand RNA viruses to initiate transcription of their genome and was first

described for Influenza virus (Plotch et al., 1981; Nguyen & Haenni, 2003). This mechanism was

investigated for TSWV by extensive inplanta studies (Duijsings et al., 1999; 2001), resulting in the

model for transcription initiation. It has been demonstrated that Alfalfa mosaic virus (AMV)

RNAs can be utilized by TSWV as cap donors during a mixed infection of N. benthamiana

(Duijsings et al., 1999). Furthermore, it was shown that suitable cap donors require a single base

complementarity to the ultimate or penultimate residue of the TSWV template (Duijsings et al.,

2001). It has been demonstrated that in vitro ongoing transcription of TSWV requires the presence

of reticulocyte lysate. This dependence was further investigated by testing the occurrence of

transcription in the presence of two translation inhibitors: edeine, an inhibitor that still allows scanning

of nascent mRNAs by the 40S ribosomal subunit, and cycloheximide, an inhibitor that completely

blocks translation including ribosome scanning. Neither of these inhibitors blocked TSWV

transcription initiation or elongation in vitro, as demonstrated by de novo-synthesized viral

mRNAs with globin mRNA-derived leader sequences, suggesting that TSWV transcription in

vitro requires the presence of (a component within) reticulocyte lysate, rather than a viral protein

resulting from translation (van Knippenberg et al., 2004).

22

2.7 THRIPS – VECTORS OF TOSPOVIRUSES

2.7.1 Transmission of viruses by thrips

Cell walls present in plants are major barrier to viral infection. Some plant viruses

depend upon vector (vehicles) for their movement from infected host plants to healthy

ones. Insects (thrips) play a very important role as vector in this viral transmission. Thrips

are important members of the ecosystems as herbivores and predators. Thrips are minute

slender bodied insects that have ability to transmit plant viruses. They are categorized as

important agricultural pest. Thrips are sap sucking insects, belong to the insect order

Thysanoptera and tospoviruses are transmitted by several species of thrips in a circulative

and propagative manner (Mound,1996; Ullman et al., 1997; Whitfield et al., 2005; Pappu

et al., 2009). Tospoviruses are not transmitted by aphids, whiteflies and leafhoppers. Till

now, there are 5000 species of thrips reported, but only 10 are known vectors of

tospoviruses (Mound, 2002; Pappu et al., 2009). But recently, a report showed that so far

14 thrips species belonging to five genera of family Thripidae, have been reported as

vectors of tospoviruses (Table 2). Genus Frankliniella including eight (8) vector species,

genus Thrips including three (3) vector species, genus Scirtothrips including one (1)

vector species, genus Dictyothrips having one (1) vector species and genus

Ceratothripoides having one (1) vector species (Jones, 2005; Whitfield et al., 2005;

Persley et al., 2006; Riley et al., 2011). Out of 14 species of thrips only five (Thrips

palmi, Thrips tabaci, Scirtothrips dorsalis, Frankliniella schultzei and Ceratothripoides

claratris) have been reported from India (Mandal et al., 2012). There was ample evidence

that the virus-vector relationships linking tospoviruses to their thrips vectors demonstrate a

high level of specificity, which also determines vector competence (Wijkamp et al., 1995;

Cabrera-La Rosa and Kennedy, 2007; Riley et al., 2011). The main TSWV vector species

all belong to family Thripidae. The main vector for the transmission of TSWV was

reported to be Frankliniella occidentais. Interaction of both these has studied as model

system to understand the basic mechanism of virus transmission. Thrips feed on plant

tissue with piercing and sucking mouth parts. Thrips acquire TSWV in two larval stages

and it is only when larvae feed on infected plant host. Once thrips acquire the virus they

remain viruliferous (infected with virus) throughout their life span. Virus acquired by the

larvae renders the thrips infectious, and transmission of the virus is mainly ascribed to

adults (Sakimura, 1962). It has been studied that TSWV infection alters the feeding

23

behavior of its insect vector. Data reveals that viruliferous males are good feeder as

compare to non- viruliferous males and for females no change was observed for their

behavior (Stafford et al., 2010). Wijkamp et al. (1993) showed that larvae of

Frankliniella occidentalis also transmit the virus efficiently. The virus upon acquisition

was shown to move through the midgut and subsequently reaches the salivary glands. It

was hypothesized that the close proximity of midgut and salivary glands in the thrips

larval stage facilitates the virus movement whereas the virus fails to do so as the thrips

reaches adult stage. This may explain the inability of adult thrips to transmit the virus if

the virus is acquired for the first time in its adult life (Filtho Assisde et al., 2004). The

specificity of TSWV and thrips vectors may be due to the presence of a receptor in the

vector species which may be absent in non-vector species. F. occidentalis and T. tabaci

have been found to be vectors of at least four important plant virus groups including the

bunyaviruses (Ullman et al., 1997). Earlier, eight species of thrips were reported to

transmit TSWV (Wijkamp et al., 1995). F. occidentalis Pergabnde (the western flower

thrips), Thrips tabaci Lindeman (the onion thrips), T. palmi Karny (melon thrips), T.

setosus Moulton, Frankliniella schultzei Trybom (the common blossom or cotton bud

thrips), F. intonsa Trybom and F. fusca Hinds (the tobacco thrips) were reported to be

vector of TSWV (Wijkamp et al., 1995; Ullman et al., 1997). Webb et al.(1997) also

reported F. bispinosa Morgan (Florida flower thrips) as a vector of TSWV. Frankliniella

tenuicornis Uzel (European grass thrips) and Scirtothrips dorsalis Hood (the chilli thrips)

had been previously reported to be vector of TSWV, but experimental verification had

not been done for all species as has been done for F.occidentalis and T. tabaci (Ullman et

al., 1997).So, F. occidentalis and T. tabaci are common and important vectors of multiple

plant viruses in many regions of the world (Ullman et al., 1997). In Thailand, C. claratris

has been reported as vector of CaCV (Premachandra et al., 2005) and in Israel, T. tabaci

was reported vector for IYSV (Gera et al., 1998). T. tabaci was the only reported

vector of IYSV (Cortes et al., 1998; Kritzman et al., 2001), but recently F. fusca has

been described as a second vector in the USA (Srinavasan et al., 2012). In Taiwan,

Peanut chlorotic fan-spot virus (PCFV) was transmitted by S. dorsalis (Chen and Chiu,

1996). F. schultzei and T. palmi have also been reported to be vector of CaCV (Persley et

al., 2006; Chiemsombat et al., 2008). In India, T. palmi was said to be suspected vector

for GBNV and WBNV (Lakshmi et al., 1995; Singh et al., 1996; Mandal et al., 2012). S.

dorsalis was found to be vector for GBNV in tomato (Meena et al., 2005) and it was also

24

reported as vector for Peanut yellow spot virus (PYSV) (Reddy, 1989). GBNV was

transmitted by Frankliniella occidentalis, F. schultzei, F. fusca, Thrips tabaci, Thrips

palmi and Scirtothrips dorsalis, but T. Palmi was the main vector of GBNV in India

(Vijay Lakshmi, 1994).

2.7.2 Mechanism of transmission

Insect vectors play a key role in dissemination of viruses that cause important

diseases in humans, animals and plants (Ullman et al., 2005). With the discovery that

TSWV multiplies in insect vectors, the complex nature of the interplay between thrips,

tospoviruses and their plant hosts was first recognized (Ullman et al., 1993; Wijkamp et

al., 1993). TSWV is able to infect both its botanical hosts and its insect vector (thrips). It

has been demonstrated by using the approach of Reddy and Black, 1966 that Tospovirus

multiply in their insect vector (Frankliniella occidentalis). The evidence that genetic

determinants for TSWV transmissibility reside on middle RNA which encode viral

glycoproteins came by creating reassortants by co inoculating plants with thrips-

transmissible isolate (TSWV-RG2) and a thrips-nontransmissible TSWV isolate (TSWV-

D) (Sin et al., 2005). Insect inoculation of tospoviruses into their plant hosts cannot

occur without viral passage across at least three insect organs (the midgut, visceral

muscle cells and salivary glands) that include six membrane barriers (Whitfield et al.,

2005). Replication of the TSWV in midgut, its movement from midgut to visceral

muscles and then the salivary glands are crucial factor determining the vector

competency (Nagata et al., 2002). Several lines of experiments demonstrated that TSWV

GPs bind to the insect midgut during TSWV acquisition by thrips and plays a critical role

in TSWV transmission by thrips. While TSWV G(N) binds the insect vector midgut and

inhibits TSWV acquisition (Ullman et al., 2005). Whitfield et al. (2008) also reported

that soluble form of the envelope glycoprotein GN (GN-S) specifically bound to thrips

midguts and reduced the amount of detectable virus inside midgut tissues. It has been

seen that increase in the concentration of two TSWV encoded proteins (N and NSs), by S

RNA firmly demonstrate replication of TSWV in its vector. The accumulation of N

protein is indicative of the production of virus particles, but the accumulation of the NSs

protein, which has not been found in virus particles (Kormelink et al., 1991) can only

occur after transcription of its mRNA from the complementary viral RNA strand which

was formed during the replication of viral RNA. Hence, the presence and increase of this

25

protein both give conclusive proof that TSWV replicates in its vector (Frankliniella

occidentalis) (Wijkamp et al., 1993). Screening of a cDNA library of F. occidentalis

using fragments of TSWV RdRp, a F. occidentalis putative transcription factor (FoTF)

was identified that binds to TSWV RdRp, which was shown to bind to TSWV RNA and

enhance TSWV replication (in vitro). Mammalian cells expressing this putative

transcription factor supported TSWV replication. So, this factor which supports TSWV

replication in vivo and in vitro could be used to compare molecular defense mechanisms

in plant, insect and mammalian cell lines against the same pathogen for better

understanding of evolutionary studies (Medeiros et al., 2005).

2.8 DETECTION AND CHARACTERIZATION

Methods that are based on serology and molecular approaches are increasingly being used to

detect plant pathogens specially viruses. These include immunological (serological) and nucleic

acid based techniques. TSWV has certain unique biological properties that are useful for

diagnosis. A turning point in TSWV detection and diagnosis came with the production of high

quality polyclonal antisera and development of an enzyme-linked immunosorbent assay (ELISA)

(Gonsalves and Trujillo, 1986). Adam et al. (1995) have described an assay which could detect

tospoviruses generally, based on antibodies to the G proteins of the virus. Dot-blot

immunoassay and tissue-print immunoassay has also been used for specific detection of

TSWV (Hsu & Lawson, 1991; Louro 1995). Serological identification was a well established

method that has been routinely used for the detection of tospovirus infection in various types of

plants (Vaira et al., 1996; Lin et al., 2005; Chen et al., 2010). However, ELISA can only be

applied where species-specific monoclonal or polyclonal antibodies are present. Therefore,

molecular biology based systems are very promising in this regard. Several PCR-based methods

have also been developed for the specific detection of TSWV. The first PCR-based assay was

developed by Mumford et al. (1994). Immunocapture PCR and RT-PCR were developed by

Nolasco et al. (1993) and Weekes et al. (1996), respectively. A lot of methods like Reverse

transcriptase polymerase chain reaction (RT-PCR), RT-PCR restriction fragment length

polymorphism (RT-PCR-RFLP) and real-time RT-PCR techniques have been used by

various scientists throughout world for identification of tospovirus infection (Roberts et al.,

2000; Chu et al., 2001; Okuda and Hanada, 2001; Uga and Tsuda, 2005; Kuwabara et al.,

2010; Debreczeni et al., 2011). Nevertheless, each of these methods also comes with its own

limitations.

26

Table 2.2: Thrips (vectors) identified for various tospoviruses.

S. No Tospovirus (Abbreviation) Vector species References

1. Alstroemeria necrotic streak virus (ANSV) Frankliniella occidentalis Hassani-Mehraban et al., 2010

2. Bean necrotic mosaic virus (BeNMV) Unknown de Oliveira et al., 2011

3. Calla lily chlorotic spot virus (CCSV) Thrips palmi Chen et al., 2005

4. Capsicum chlorosis virus (CaCV) Ceratothripoides claratis Premachandra et al., 2005

T. palmi , F. schultzei Chiemsombat et al., 2008; Persley et al., 2006

5. Chrysanthemum stem necrosis virus (CSNV) F. occidentalis, F. schultzei Bezzera et al., 1999; Nagata and de Ävila, 2000; Nagata et

al., 2004

6. Groundnut bud necrosis virus

(Peanut bud necrosis virus) (GBNV/PBNV)

F. schultzei, T. palmi,

S. dorsalis

Amin et al., 1981; Lakshmi et al., 1995; Meena et al.,

2005

7. Groundnut chlorotic fan-spot virus (GCFV) S. dorsalis Chen and Chiu, 1996; Chu et al., 2001

8. Groundnut ringspot virus (GRSV) F. occidentalis Wijkamp et al., 1995

F. intonsa, F. fusca Nagata et al., 2004; De Borbon et al., 2006

9. Groundnut yellow spot virus (GYSV) S. dorsalis Reddy et al., 1991; Gopal et al., 2010

10. Impatiens necrotic spot virus (INSV) F. occidentalis Wijkamp et al., 1995

F. intonsa, F. fusca Sakurai et al., 2004; Naidu et al., 2001

11. Iris yellow spot virus (IYSV) T. tabaci, F. fusca Cortes et al., 1998; Srinivasan et al., 2012

12. Melon yellow spot virus (MYSV) T. palmi Kato et al., 2000

13. Peanut chlorotic fan-spot virus (PCFV) S. dorsalis Chen and Chiu, 1996

14. Pepper necrotic spot virus (PNSV) Unknown

15. Polygonum ring spot virus (PolRSV) Dictyothrips betae Ciuffo et al., 2010

16. Soybean vein necrosis- associated virus (SVNaV) Unknown Zhou et al., 2011

17. Tomato chlorotic spot virus (TCSV) F. occidentalis, F. schultzei, F. intonsa Wijkamp et al., 1995

18. Tomato necrotic ringspot virus (TNRV) C. claratis, T. palmi Seepiban et al., 2011

19. Tomato yellow ring virus (TYRV) T. tabaci Rasoulpour and Izadpanah, 2007

20. Tomato zonate spot virus (TZSV) Unknown

21. Tomato spotted wilt virus (TSWV) T. tabaci, F. occidentalis, Wijkamp et al., 1995

F schultzei, F. intonsa Avila et al., 2006

F. bispinosa ,F. cephalica Ohnishi et al., 2006, Sakimura,1963

F. fusca, F. gemina De Borbon et al., 2006; Fujisawa et al., 1988

T. setosus, T. palmi Persley et al 2006

22. Watermelon bud necrosis virus (WBNV) T. palmi Jain et al., 1998; Pappu et al., 2009

23. Watermelon silver mottle virus (WSMoV) T. palmi Yeh et al., 1992; Chiemsobat et al., 2008

24. Zucchini lethal chlorosis virus (ZLCV) F. zucchini Nakahara and Monterio, 1999

27

TSWV detection by molecular methods has also been developed using cDNA probes

(Ronco et al., 1989; Rice et al., 1990) and riboprobes (Huguenot et al., 1990), both of which

have proved useful for the sanitary certification of plant material (Saldarelli et al., 1996). A very

sensitive protocol for the detection and quantification of TSWV was known as the real-time RT-

PCR assay based on TaqManTM

chemistry, on both “leaf soak” and total RNA extracts from

infected plants (Roberts et al., 2000). A comparison between ELISA and RT-PCR assays was

done to detect TSWV in field-grown chrysanthemum (Matsuura et al., 2002; 2004) and recently for

peanut (Dang et al., 2009). Similarly potential of RT- PCR was evaluated by comparing its

sensitivity with DAS-ELISA for the detection of TSWV among 22 Australian plant species.

DAS-ELISA was found to be less sensitive as compare to RT-PCR method (Dietzgen et al.,

2005). One-step multiplex reverse transcription-polymerase chain reaction (multi-PCR) was also

utilized for simultaneous identification of five tospovirus species (Kuwbara et al., 2010). For the

detection of TSWV in individual thrips, a sensitive and robust real time fluorescent (RT-PCR

Taqman) technique was developed (Boonham et al., 2002). Similarly, by using RT-PCR

technique TSWV was detected from a single infected thrip (Mason et al., 2003). A quantitative

real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general

primer set and three TaqMan(®)MGB probes was developed for general and genotype-specific

detection and quantization of the genomic M segment of TSWV (Debreczeni et al., 2011). All the

techniques described have their own limitations. Recently, a study reports the development of

multiplex RT-PCR-ELISA for the detection and identification of four tospoviruses species Melon

yellow spot virus (MYSV), Tomato necrotic ringspot virus (TNRV), Watermelon silver mottle virus

(WSMoV), and Capsicum chlorosis virus (CaCV) (Charoenvilaisiri et al., 2014).

2.9 IMMUNOSENSORS (BIOSENSORS)

There was a noteworthy trend towards development of fast, efficient and cost effective

techniques for the detection and quantification of various diseases. There is increasing need for

research that includes development of tools that are sensitive, quicker as compare to traditional

available techniques. Traditional methods for virus detection are Enzyme linked immunosorbent

assay (ELISA) and Reverse Transcriptase -Polymerase Chain Reaction (RT-PCR) were time

consuming and expensive and Biosensor showed promise in this regard. Biosensor is an

analytical device that converts biological reaction into measurable signal which is proportional to

28

analyte concentration. They consist of biological sensing element and a transducer for the detection

of analyte concentration. Sensing element includes microorganism (whole cell biosensors),

enzymes, antibodies (Immunosensors) and biological tissues and organelles. On the basis of

transducer they can be classified as electrochemical (Amperometric, conductometric and

potentiometric), optical (absorbance, fluorescence, and chemiluminesence), Piezoelectric (Acoustic

and ultrasound) and colorimetric (Coreurea and Calvalieri, 2003). Transducer is an analytical tool

which provides an output quantity having a given relationship to input quantity (McNaught and

Wilkinson, 1997). Biosensors offer several advantages in comparison to many conventional

analytical approaches in terms of simplicity, lower limit of detection and sensitivity. One of the

greatest advantages is that Biosensors enjoy specificity due to their exploitation of biological

molecules such as enzymes or antibodies. Biosensors based on many technologies have wide range

of applications, detecting plant viruses in agriculture is one of such applications (Vashpanov et al.,

2008). However, a very few biosensor have been commercialized in this direction (Vashpanov et

al., 2008).

Amperometric biosensor has been used on large scale for analyte such as glucose and lactate

(Ohnuki et al., 2007), and sialic acid (Marzouk et al., 2007). They were also used in the detection of

pesticides and nerve gas (Liu and Lin, 2006). Bacillus cereus and Mycobacterium smegmatis have

been detected by using amperometric biosensor (Yemini et al., 2007). It has also been used in

serological diagnosis of Francisella tularensis (Pohanka and Skladal, 2007a). Pharmacology study

has also been described by use of amperometric biosensor (Pohanka et al., 2007c). Measurement of

analyte such as phenol has also been described using Pesudomonas species (Skladal et al., 2002).

Enzyme based biosensor for the rapid detection of organophosphates and carbamates have also been

worked out (Skladal, 1996). Similarly, microbial sensor for measurement of ammonia and methane

has been studied (Wittman et al., 1997). Amperometric biosensor was also evaluated for assays with

nucleic acid acting as a marker and/or biorecognition component; uropathogens were assayed using

their 16S rRNA (Liao et al., 2006). Glucose biosensor was a well known commercial amperometric

biosensor. The device Medas Pro was employed for the analysis of surface water (Rosseti et al.,

2001). In case of Immunosensors, unique property of antibody was utilized where antigen fits into

specific antibody binding site (Vo- Dinh and Cullum, 2000). First type of DNA biosensor used for

the detection of plant viruses was Quartz Crystal Microbalance (QCM) (Eun et al., 2002). It was

used for the detection of two plant viruses Cymbidium mosaic virus (CymMV) and Odontoglossum

29

ring spot virus (ORSV) from different groups namely potexvirus and tobamovirus respectively.

Recently, there was a report showing use of cell biosensor for detection of plant viruses (Perdikanis

et al., 2011). The sensor was based on the change of the membrane electric potential of a host cell

during the interaction with corresponding virus particles. This BERA-HTP (High Throughput

Bioelectrical recognition Assay) was tested for the identification of Cucumber mosaic virus (CMV),

Tobacco rattle virus (TRV), and Potato virus Y (PVY). Similarly, modified mesoporous silicon

electronic device has been used to detect biological particles having less then 50nm diameter. They

have been used to detect biological virus particles such as Tomato ring spot virus (ToRSV) and

Gravevine fan leaf virus (GFLV) (Vashpanov et al., 2008). Immunosensor have also been used for

the detection of Plum pox virus (PPV) and Prunus necrotic ringspot virus (PNRSV) affecting stone

fruits and doing great economic losses (Radecka et al., 2013).