REVIEW AND LITERATUREshodhganga.inflibnet.ac.in/bitstream/10603/44834/2... · 2.2 Details of...

Review and Literature

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REVIEW AND LITERATURE

2.1 Petunia

Petunia (Petunia hybrida Hort.) belongs to the family Solanaceae and is an economically

important plant (Christopher 1994; Davies et al. 1998). It is originated from America, and is a

widely cultivated plant used in flower beds, hanging baskets, window boxes and other types of

containers and as cut flowers. Most of the varieties of P. hybrida are grown in gardens

throughout the world for their attractive beautiful blooms of various colors (Winterrowd 2004).

Besides its floriculture importance, it is an important source of anti-microbial agent (Rahman et

al. 2008) and for anti-oxidation activity.

2.1.1 Morphological details of petunia

Petunias are compact, erect type of plants, reaching 15 to 25 cm and adapted for summer garden

beds, and the sprawling, long-stemmed balcony petunia, which grows to about 45cm. Leaves are

alternate, broad-ovate to cordate shaped soft, flabby, and covered with fine, sticky hairs and

length is 4 cm to 8cm long. Flowers are funnel-shaped (tube with a very broad limb from 5cm to

12cm) corolla and form, single or double, solid or bicolor, fragrant. It is greatly diversified and

available in a range of colors (Christopher 1994).

2.1.2 Genus and species of petunia

In 1803, Jussieu established the genus Petunia (Solanaceae) and name Petunia derived from

French, which took the word petun, meaning "tobacco," from a Tupi–Guarani language. To

date, around 35 Petunia (sub) species have been described. Two important species of petunia are

P. axillaris and P. integrifolia.

P. axillaris: P. axillaris bears night-fragrant, buff-white blossoms with long, thin tubes and

somewhat flattened openings and is an annual herbaceous plant. The species was first sent from

South America to Paris in 1823.

P. integrifolia: P. integrifolia has a somewhat weedy habit, spreading stems with upright tips,

and small lavender to purple flowers. It was discovered in South America by the explorer James


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Tweedie, after whom the genus Tweedia is named, who sent specimens to the Glasgow

Botanical Garden in 1831.

P. × hybrid is derived from a cross between P. axillaris and P. integrifolia (Stehmann et al.

2009). Most of the varieties grown in gardens are Petunia hybrida (Winterrowd 2004).

2.1.3 Climate and soil for petunia cultivation

Petunia seeds germinate in 5 to 15 days. Petunias can tolerate relatively harsh conditions and hot

climates. They need at least five hours of sunlight every day. They grow well in low humidity,

moist soil. Young plants can be grown from seeds. Petunias should be watered once every two

to five days. In drier regions, the plants should be watered daily. Dead petals should be pruned

so that the younger branches can flourish. Maximum growth occurs in late spring. Applying

fertilizers once a month will help the plant grow quickly. Blooming season of petunia is spring

to fall with decline in summer, frost tolerant; may consider using for an early spring flowering

show (February to May) as it is frost tolerant.

2.1.4 Origin and distribution: The geographic origin of Petunia is the southern/central

part of South America, and various species have been documented from collections made in

Argentina, Brazil, Paraguay, and Uruguay (Ando et al. 2005). Now, petunia is distributed

worldwide.

2.1.5 Economic Importance

Ornamental plants: Petunia is a widely cultivated ornamental plant used in flower beds,

hanging baskets, window boxes and other types of containers and as cut flowers. In 2008,

wholesale value of petunia is $110 million in United States (U. S. Department of Agriculture–

National Agricultural Statistics Service, 2009).

Antimicrobial agent: Besides its floriculture importance, it is an important source of an anti-

microbial agent (Rahman et al. 2008) and also showed the mildest anti-oxidation activity. Its

leaves yield an important insecticide widely used against a broad range of insects.


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Valuable model system for studies of gene function: Petunia has played a central role in

transformation research as many reports on plant transformation exist in literature by earlier

researchers which are described in table 1. It was considered as a key model system when the

first definitive account of Agrobacterium-mediated transformation and direct DNA transfer

unequivocally was established for the transfer and expression of foreign genes in plants. One of

the key reasons for the importance of petunia in plant transformation research has been the

selection for and/or identification of genotypes well suited to growth and regeneration in culture,

Agro-infiltration for transient gene expression and the development of intragenic vectors to

affect gene transfer without the integration of “foreign” DNA represent recent advancements in

petunia transformation.

Table 1 Some important gene has been integrated in petunia genome for gene function analysis.

Foreign genes integrated in petunia genome References

Pokeweed antiviral protein (mutant PAP) Li et al. 2013

CYP2E1 Zhang et al. 2011

Isopentenyl transferase(Ipt gene) Bai et al. 2009

Cistran of PVY (Protease-Replicase-Coat protein) Ziv et al. 2005

Ultra Blue Delta-9, fatty acid desaturase Choudhury et al. 1994

Tryptophan decarboxylase [aromatic L-amino acid decarboxylase] Thomas et al. 1999

GUS (uid A) naringenin-chalcone synthase (GTCHSI) Kobayashi et al. 1998

2.1.6 Viruses/viroids/phytoplasma pathogens affecting petunia cultivation

The cultivation of petunia has been affected due to several viral pathogens which cause

considerable economic losses to the petunia. Some of them are: Potato virus Y, Tobacco mosaic

virus, Tomato mosaic virus, Alfalfa mosaic virus, Cucumber mosaic virus, Petunia vein clearing

virus and Broad bean wilt I virus (Lesemann 1996; Mavric et al. 1996; Cohen and Sikron 1999;

Alexandre et al. 2000; Liu et al. 2003; Sabanadzovic et al. 2008). Besides viral diseases, viroid

and phytoplasma diseases are also found affecting petunia. Some of the diseases caused by

viruses, viroids and phytoplasma in petunia have been summarized in table 2.


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2.2 Details of Gerbera

Gerbera, African daisy, is a genus of ornamental plants from the sunflower family

Asteraceae. Gerbera is very popular and widely used as a decorative garden plant or as cut

flowers of the wide range of colors include yellow, orange, cream, white, pink red, scarlet,

salmon, maroon, terracotta and various other intermediate shades. Gerbera is also important

commercially. It is the fifth most used cut flower in the world (after rose, carnation,

chrysanthemum and tulip) (http://en.wikipedia.org/wiki/Gerbera).

2.2.1 Morphological details

Plants are stem-less, tender and perennial herbs. They are dwarf, 30-45 cm tall & hairy

thought. The leaves are 12-20 cm long, 5-7 cm broad, deeply lobed with a fairly long (15

cm) petiole. Leaves are leathery, narrower at the base and wider at the top and are arranged

in rosette at the base, the foliose in some species is entire and has light under surface. The

flowers are daisy like, 7-10 cm across but in certain hybrids these may be as large as 15 cm

across. Flower head solitary, many flowered the conspicuous ray in 1 or 2 rows, those are the

inner row, when present, very sort and sub tubular and two-lipped. The flower may be single

or double and available in various self colored cultivar as well as bicolours. During recent

year, the breeders devolved many varieties, the florets of which are Known as, Black hearts,

or Green hearts on the basis of color of disc. There are double and bicolour varieties also.

Flower stalk are long, slender and leafless. Achenes are beaked and pappus.

2.2.2 Species and cultivars

The genus Gerbera consists of about 40 species of half hardy and perennial flowering plants.

There are about 30 species, native to Africa, Madasagar, and Asia. In Southern Africa, there

are 13 native species (Hansen 1999). Out of the recorded species, only one species G.

jamesonii is under cultivation. Gerbera is diploid (2n = 50) and basic chromosome number is

n = 25.

Some important species of gerbera

Gerbera asplenuifolia: Leaves narrow, 10-15 cm long, more or less deeply lobed, leathery,

glossy above, loves roundish, concave margines, revolute flower head purple on a hairy

scape.


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Gerbera aurantiaca (Hilton Daisy): Leaves are lanceolate to oblong, acute 12.5-15cm long,

entire or toothed flower heads are orange and anther yellow.

Gerbera jamesonii (Barberton Daisy): Hairy throughout, Base woody, leaves lobed, many

solitatary, orange, scarlet, heads 7.5-12.5 cm or more across, are borne from May to August.

Single or double flowered cultivars and hybrids in attractive colours are generally available.

Gerbera kunzeana: A Himalayan species whose flowers scarcely open.

Gerbera viridifolia: Leaves elliptical or oblong, obtuse, green on both sides and smooth or

nearly so. Flower stalks short. Flower heads dirty white and small.

Gerbera hinlonnii: Hapatalia hintonii, endemic to Mexico, has floral characters that

indicate affinities to the genus Gerbera and new nomenclature Gerbera hintonii is proposed

(Katinas 1998).

Gerbera maxima (D. Don) Beauv: A rare plant rediscovered after a century from Pauri

Garwal, India.

The domesticated cultivars G. hybrida are mostly a result of a cross between G.

jamesonii and another South African species G. viridifolia and because of these genetic cross

thousands of cultivars exist.

Gerbera cultivars of commercial importance throughout the world: Zingaro (red),

Silvester (white), Solvadore (yellow), Rosalin (pink), Davaellen, Goliath, Creem Climentine

(creemy white), Maron Clementine (orange), Flamingo (Pale rose), Delphi (white), Vesta

(red), Urenus (yellow), Fredenking (yellow), Terra queen (Pink), Dustty (red), Valentine

(pink), Labalga (lilac), Fredaisy (pink), Fredorella (red) etc.

There are many other cultivars which are cultivated commercially in India are:

Cream Clementine, Maron Clementine, Delphi, Vesta, Uranus, Terraqueen, Dusty,

Valentine, Diablo, Mariso and Pascal.

2.2.3 Origin and distribution of gerbera

In 1880, Captain Robert Jameson, a Scotsman, first discovered gerbera daisies while

operating a gold mine near Barberton in the Transvaal area of South Africa and took it with

him to Great Britain. He donated plants to the Durban Botanical Gardens, and the curator of


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the gardens, John Medley Wood, sent specimens to Harry Bolus in Cape Town, South

Africa, for identification. Bolus then sent specimens to the Royal Botanical Gardens at Kew

in England. The first scientific description of a gerbera was made by J.D. Hooker in Curtis's

Botanical Magazine in 1889 when he described G. jamesonii, a South African species. The

genus Gerbera was named in honor of a German botanist and naturalist, Trangott Gerber

who traveled in Russia in 1743 and was a friend of Carolus Linnaeus. This genus is native to

South African and Asiatic regions. They mostly inhabit temperate or mountainous regions.

The native distribution of this genus comprising 30 species extends to Africa, Madagaskar,

Tropical Asia and South America. It is grown in a variety of climates in all parts of the

world. According to Dr. Yoseph Shoub (Gerbera Breeding Ltd. Israel), Subtropical climate

and Mediterranean climate is suitable for growth as cut flower around the world. These

climate zones pass through Israel, Italy, Spain, Portugal, Morocco, Colombia, Japan, South

Africa, Australia and Southern India.

Fig. 1 The red colored areas in the world map are the major gerbera growing area.

Although the varieties are best suitable to these conditions, they do very well in other

climate as well. The most important production areas are: the Netherlands, Italy, Germany,

France, and California. About 7 species were recorded in India, distributed in the temperate

Himalayas from Kashmir to Nepal at altitudes of 1,300 to 3,200 meters.

2.2.4 Soil and climatic conditions

Soil: Warm, sandy or organic and fast draining. In landscape bedding situations, soils must

be heavily amended with organic compost sand.

Temperature: The optimal growing temperature depends on the light intensity and time of

year. For the different seasons the following optimum temperatures can be recommended.


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Table 3 Optimum temperatures recommended for growing gerbera in different seasons.

Season Av. Day Av. Day Temp (ºC) Av. Night Temp (ºC)

Summer 24-26 18-20

Fall 21-24 16-18

Winter 19-22 14-16

Spring 21-24 16-18

The average daily temperature for maintaining sufficient production is 16ºC. The

minimum temperature for having production is 7ºC. Temperatures below 7ºC stop the

formation of buds and the chances of root diseases increase. Freezing temperatures (below

0ºC) will cause the plants to die due to frost. Temperatures higher than 30ºC will also slow

down the formation of buds. The absolute Temperature is 42ºC; above this plant loss will

occur due to destruction of plant proteins.

Relative humidity: The optimal humidity is 70% during the day and below 85% at night

and with a higher humidity, fungus problems arise such as Botrytis and Sclerotinia. High

humidity appears to contribute to flower stalk stretching and due to low humidity level, the

temperature can be lowered, ventilation reduced or moisture can be created by misting. A

high humidity level can be prevented by ventilation and heating to transport the moisture.

So, Good internal air circulation in the greenhouse at night and ventilation during the day are

essential.

Light: Gerberas require high light intensities for good-quality plants and high flower bud

numbers. For this reason, they are mostly produced in the spring and summer, with the

greatest amount of production for the spring market. Plants grow best in full sun during the

fall, winter, and spring. Light shade (30 to 40 percent) can be used to reduce excessive

greenhouse temperatures in the summer. Plants receiving too little light have pale green,

stretched foliage and long, weak flower stems. Plants receiving too much light have

compact, slightly yellow foliage with short flower stems often hidden in the foliage.

Gerberas appear to respond only slightly to photoperiod. Short days tend to speed flower

production, while long days delay flowering.

Watering: Gerberas should receive a thorough watering and then be allowed to dry

somewhat. This limits growth of the flower stem and discourages soilborne diseases.

Gerberas should never be allowed to wilt, however. Plants allowed drying out too much and


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too frequently having short flower stems that may be hidden in the foliage. It is also a good

practice to water early in the day so the foliage is completely dry before evening.

Plant spacing: After 4 weeks pot-to-pot, space the plants so that plenty of light reaches the

crown and there is free air movement. Tight spacing can delay flowering and cause leaves to

stretch. Exact spacing is difficult to recommend because of differences in pot size and

cultivar sizes. However, begin with a 6- by 6-inch minimum for 4- to 4-1/2-inch pots, an 8-

by 8-inch minimum for 5-inch pots, and a 10- by 10-inch minimum for 6-inch pots.

Crop scheduling: The timing of Gerbera daisy crops depends on several factors including

cultivar, environmental conditions, pot size, and cultural practices. Growers should keep

detailed records of crop performance and timing to improve future scheduling efforts.

Generally, 4-inch pots require 8 to 11 weeks, 5-inch pots require 9 to 12 weeks, and 6-inch

pots require 10 to 13 weeks from transplanting to finish in the summer. An additional week

is required during the winter. A general outline of gerbera daisy crop scheduling is shown in

figure 2.

14 days

14 days

4 to 6weeks4 weeks2 week

Sow Germi-nation

2 to 4 weeks

6 -8 weeks 6 -10 weeks

Largerflat

FinishPot-to-PotFinalcontainer

Fig. 2 Gerbera Production Schedule

Yield: The crop yields 2 stems / plant / month. Harvest starts from 3rd month of planting

and continued up to two years. Under open condition, 130 -160 flowers / m2 / year and

under greenhouse condition, 175 - 200 flowers /m2 / year can be obtained.

2.2.5 Production of gerbera all over the world

Gerbera is a very attractive, commercial cut flower crop and marketed in the International

florists’ trade in huge quantities. These plants are grown throughout the world in a wide

range of climatic conditions. The Netherlands produces 420 million stems of gerbera per


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year which is valued at 145 million Netherlands guilders (Sudhagar 2013). The production of

gerbera was approximately US$ 220 million in 2001 representing 70 million stems sold in

US alone (Broek et al. 2004). The crop’s commercialization continued to spread throughout

the world and, by the 1940s, there were producers in Italy, Germany, The Netherlands, New

Zealand, South Africa and United State

2.2.6 Production and cultivation of gerbera in India

According to DBT and Small Farmer’s Agribusiness Consortium, India, gerbera ranks as 2nd

most domestic tissue culture flower crops in India after carnation, anthurium, orchids in

floriculture industry. The cut flower sticks of gerbera are been sold in market with the

variable rate depends on the flower quality and size. According to recent survey of

floriculture today, gerbera farmers have recorded earnings of Rs. 25,000 - 30,000/- from an

area of 134 sqm. The average income per unit area perhaps is the highest in floriculture,

ranging from Rs. 100 to Rs. 200 per sqm. Gerbera production is maximum (2016 MT in 70

ha area) among the others cultivated flowers such as carnation, gladiolus, marigold, rose

tuberose in Uttrakhand. Gerbera cultivation in various states of India is shown in figure 3.

Aruna cha l Pra desh

Jammu K ashmir

Nagaland

Gujarat

G oa A nd hra Pradesh

HaryanaN ew D elhi Sikkim

Karnataka

Chhattisgarh

Uttrakhand

Major gerbera producing state in India

Fig. 3 Map showing the major gerbera producing areas in India. Map was drawn on the basis

of database obtained from National Horticulture Board (NHB) of India in 2011-13.


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According to National Horticulture Board (NHB) of India 2011-12 and 2012-13,

major gerbera producing states are Karnataka, Arunachal Pradesh, Sikkim, Haryana Jammu

and Kashmir, Haryana and Nagaland (Table 4).

Table 4 India top most states producing gerbera and there production during 2011-2013.

S. No. States 2011-12 (in 1000 Tones) 2012-13 (in 1000 Tones) 1 Karnataka 297.00 - 2 Arunachal Pradesh - 88.00 3 Sikkim 16.58 17.00 4 Jammu & Kashmir 3.00 5.00 5 Haryana - 2.40 6 Nagaland 3.24 0.40 Total 319.82 112.80

Source: National Horticulture Board (NHB).

The area under gerbera cultivation in Karnataka is estimated at 25 ha with production of 53

lakhs cut flowers at an estimated value of Rs. 15 lakh.

2.2.7 Estimation of gerbera production in polyhouse

The cut-flower farmer was having single polyhouse of gerbera on 0.1 hectare of land, the

cost of cultivation and gross return of gerbera was found to be Rs. 10, 26,740 and Rs. 11,

72,466 for ‘first year’. Net profit from gerbera cultivation was Rs. 1, 45,726. Output-input

ratio was 1.14 and per box cost of cultivation of gerbera was Rs. 1,711.23 for first year

(Bhosale et al. 2011)

2.2.8 Economic Importance of gerbera

Gerbera is known as an important commercial flower crop and considered as the most

important plant in the world, together with the rose, chrysanthemum, carnation and tulip.

Gerbera is amongst the ten most important commercially grown flower crop in the world.

Gerbera is highly suitable for beds, borders, pots and rock gardens. The wide range of color

and the attractive shape of flowers suit very well in flower arrangements. The cut boom has

long vase life. Now, is in great demand with good market price in the floral industry as cut

flower as well as potted plant due to its beauty, wide range of attractive flower colors (such

as red, pink, orange, peach, maroon etc.), long vase life, and ability to rehydrate after long

transportation which makes it a valuable ornamental species; it stands among the top ten cut

flowers of the world (Parthasarathy and Nagaraju 1999). In 1991, Gerbera was ranked sixth


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in sales through Dutch flower auctions and it is sold both as a cut flower and as pot plants.

Gerbera is grown commercially in India for export and domestic market. In India, we

produce very high quality cut flower of the crop and millions of tissue cultured plants.

Tropical Floritech Pvt. Ltd. in Bangalore is the leading player in commercial cultivation in

India (Chaudhary and Prasad 2000).

2.2.9 Pathogens affecting gerbera cultivation

Gerberas have been affected by wide variety of pathogens which includes insects root-knot

nematodes, bacteria such as Pseudomonas cichorii, fungi, phytoplasma and viruses.

Table 5 List of insects, bacteria, fungi, phytoplasma and viruses infecting gerbera. Insects Whitefly Trialewiodes vaporariorum Leaf miner Liriomyza trifolii and L.soncho Mites Steneotarsonemus pallidus and Polyphagotarsonemus latus Aphid Myzus persicae and Aphis jabae

Fungi: Disease Pathogen/Cause Symptom References Alternaria Leaf Spot

Alternaria alternate

Leaf spots Farhood and Hadian 2012

Leaf Spot Corynespora cassiicola

Leaf Spot Shi et al. 2012

Downy mildew Bremia luctucae yellow discoloration on leaf, later turning light to dark brown

Wolcan et al. 2010

Powdery mildew Golovinomyces cichoracearum

White fungal growth develops on the surface of leaves

Troisi et al. 2010

Alternaria leaf spot

Alternaria spp. oval, circular or irregular, brown to black lesions with concentric rings on leaf

Mirkova and konstantantinova 2003

White rust Albugotragopogonis

white erumpent sori Vazquez et al. 1997

Bacteria: Bacterial leaf spot

Pseudomonas cichorii

Small to large spots are circular at first, and then become irregular and dark brown to black.


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Phytoplasma: Phytoplasma Country References Candidatus Phytoplasma Asteris’ Southern Italy Spano et al. 2011 Phytoplasma, 16SrII Australia Siddique et al. 2005 Phytoplasma, 16SrI Italy Bertaccini and Bellardi 1998

Viruses:

2.3 Cucumber mosaic virus (CMV)

2.3.1 Economic importance Cucumber mosaic virus (CMV) is an important virus because of its agricultural impact in the

Mediterranean Basin and worldwide, and also as a model for understanding plant–virus

interactions. CMV making a strong appearance on the basis of their scientific importance

ranked 4rth position after Tobacco mosaic virus, Tomato spotted wilt virus and Tomato

yellow leaf curl virus in Top 10 plant virus list for Molecular Plant Pathology prepared

based on more than 250 votes from the international community (Table 6).

It is perhaps justified that TMV and CMV are the two highest placed in terms of

scientific importance, as a search of the ISI WEB of Science database in 2011 for papers

with these viruses in their titles yielded counts of 3636 (TMV) and 1258 (CMV) versus

counts for the other viruses (BMV, PVX and CaMV) of 400–600 for each.

Viruses Symptoms Country References Tomato spotted wilt virus

Concentric rings and distortion of leaves

Serbia Stankovic et al. 2011

Southern Italy Spano et al. 2011

Tomato black ring virus - China Zhang et al. 2009 Impatiens necrotic spot virus

Necrotic spot on leaf New Zealand Elliott et al. 2009

Tobacco mosaic virus - China Zhang et al. 2009 Cucumber mosaic virus Color break on the

petals, and deformed flowers

India Verma et al. 2004a

Tobacco rattle virus

-

Netherland Schmelzer et al. 1966; Stouffer et al.1965


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Table 6 Top 10 plant viruses. The table represents the ranked list of plant viruses voted for

by plant virologists associated with Molecular Plant Pathology Association.

Rank Virus Author of virus description

1 Tobacco mosaic virus (TMV) Karen-Beth G. Scholthof

2 Tomato spotted wilt virus (TSWV) Scott Adkins

3 Tomato yellow leaf curl virus (TYLCV) Henryk Czosnek

4 Cucumber mosaic virus (CMV) Peter Palukaitis

5 Potato virus Y (PVY) Emmanuel Jacquot

6 Cauliflower mosaic virus (CaMV) Thomas Hohn and Barbara Hohn

7 African cassava mosaic virus (ACMV) Keith Saunders

8 Plum pox virus (PPV) Thierry Candresse

9 Brome mosaic virus (BMV) Paul Ahlquist

10 Potato virus X (PVX) Cynthia Hemenway

The table represents the ranked list of plant viruses voted for by plant virologists.

2.3.2 Taxonomic position

The 8th International Committee on Taxonomy of Viruses (ICTV) approved the placement of

Tomato aspermy virus (TAV), Cucumber mosaic virus (CMV) and Peanut stunt virus (PSV)

as the type species of genus Cucumovirus, under family Bromoviridae (van Regenmortel et

al. 2000; Fauquet et al. 2005) based on nucleic acid content (plus sense RNA genome) and

strandedness (single stranded). Family Bromoviridae contain five genera: Alfamo, Ilar,

Bromo, Cucumo and Oleavirus.

2.3.3 Brief description of CMV:

CMV is functionally described as tripartite virus containing single-stranded, plus sense

messenger RNA genome. RNA is packaged in three different icosahedral particles. Virions

are icosahedral with T=3 quasi symmetry, 29 nm in diameter without a conspicuous

capsomere arrangement (Brunt et al. 1996) and consist of 18.2% RNA (Francki and Hatta

1980). It infects over 1200 species of hosts including members of 85 plant families and

affected a great variety of ornamental, vegetable and other plants by causing severe losses in

yield and quality (Bouwen and van-Zaayen 1995; Palukaitis and Gacia Arenal 2003).


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In nature, it is transmitted by aphids in a non-persistent manner, and through the

seed in some plant hosts. Experimentally, it is easily transmitted by mechanical inoculation

of plant sap. It is distributed world-wide, causes economically important diseases in a large

variety of crop plants, and has the widest host range of any known plant virus.

CMV was first reported by Doolittle (1916) as the causal agent of disease in

cucumber and muskmelon, hence named Cucumber mosaic virus. Then after, CMV has been

reported to cause mosaic in cucumber, melon and other cucurbits; blight in spinach; mosaic,

fern leaf and systemic necrosis in tomato; mosaic and ringspot in pepper; mosaic and

stunting in clover, lupins and lucerne; stunting in soybean; mosaic, infectious chlorosis and

heart rot in banana; and mosaic and dwarfing in many other species of dicotyledonous and

monocotyledonous plants (Kaper and Waterworth 1981; Palukaitis et al. 1992; Brunt et al.

1997).

2.3.4 Some well characterized strains of CMV

Many symptom variants occur, making the virus often difficult to identify from symptoms

alone. Many strains have been differentiated on the basis of symptom and host range,

transmission, serology, physicochemical properties and nucleotide sequence. Most strains

have a restricted geographic distribution. Among the many well-characterized strains are O,

Y, Q, S, D, Ix, M, B, LS, WL, Fny, Tfn and NT9 (Plant Virus Description

http://www.dpvweb.net/dpv/showdpv.php?dpvno=400)

Recently CMV has been reported on Solenum melangena (Kumar et. al. 2014),

Petunia hybrida (Gautam et al. 2012), Risnus comunicus (Raj et al. 2010) Jatropha curcas

(Raj et al. 2008a), Rauvolfia serpentine (Raj et al. 2007), Cymbopogon citrates (Raj et al.

2007a), Catharanthus roseus (Samad et al. 2008) from India.

Various CMV strains reported recently from all over the world are summarized in

table 7.


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2.3.5 Host range and symptomatology

CMV has the widest host range for any plant virus, including more than 1200 species in over

100 families of dicotyledonous and monocotyledonous angiosperms (Edwardson and

Christie 1991). CMV induces a variety of symptoms depending on the host plant species and

cultivar, and on the virus strain. CMV induces a systemic infection in plants and the most

common symptom is a mosaic pattern (Doolittle 1916). Symptoms induced by CMV in 30

important food and ornamental crops were described by Smith (1972) which vary widely in

nature depending on the strain type. A number of other symptoms are mottling of leaves or

flowers, stunting or yellowing of entire plants, flecking, dwarfing and fern leaf (Pratap et al.

2008; Raj et al. 2008a). Some of the intermediate symptoms include blight, shoestring, ring

spot, fruit woodiness and necrosis of bulbs and fruits (Kaper et al. 1981; Raj et al. 2008b).

Besides, co-infection of CMV with some other non-related virus produces severe synergistic

effects (Tien et al. 1987; Kuhn 1990; Ryang Bo-Song et al. 2004; Xi et al. 2007; Wege and

Siegmund 2007). The RNA 5 associated with CMV are known as CARNA5 or satellite

RNAs. They are also known to have regulatory effect on symptom expression (Raj et al.

1999a). Satellite RNAs may exacerbate (Kaper and Waterworth 1977) or cause attenuation

in disease symptom (Waterworth et al. 1979). The virions of CMV are found in all parts of

the host plant in cytoplasm. The inclusions present in infected cells are crystals in the

cytoplasm (that are often rhomboidal, hexagonal or roughly spherical and may appear as

solid hollow structures) containing virions (Brunt et al. 1996).

CMV is very common in temperate regions and exists as its numerous strains that

differ in their host preference, symptoms induced, mode of transmission and some other

properties (Agrios 1978; Francki et al. 1979). CMV is known all over the world by various

synonyms depending on the host plant and the symptoms it produces for example, banana

infectious chlorosis virus, coleus mosaic virus, cowpea banding mosaic virus, cowpea ring

spot virus, cucumber virus, lily ring spot virus, pea top necrosis virus, peanut yellow mosaic

virus, soybean stunt virus, spinach blight virus, tomato fern leaf virus, pea western ring spot

virus etc (Kaper et al. 1981). Ornamental hosts of CMV are chrysanthemum, amaranths,

aster, delphinium, salvia, geranium, gilia, gladiolus, heliotrope, larkspur, lily, marigold,

morning glory, nasturtium, periwinkle, petunia, phlox, snapdragon, tulip, and zinnia (Chupp

and Sherf 1960; Agrios 1978). Other vegetable hosts are muskmelon, squash, peppers,

turnip, watermelon, pumpkin, broad bean, onion, eggplant, potato, carrot, parsley (Chupp

and Sherf 1960), loofah (Huang et al. 1987), artichoke (Chabbouh and Cherif, 1990) etc.


23

Propagation species

Nicotiana tabacum or N. glutinosa are convenient for maintaining cultures. Cucurbita spp.

and N. tabacum are suitable as virus source plants for purifying the virus, but N. clevelandii

appears the best source for most strains.

Diagnostic host species: The common diagnostic for CMV and there symptom describe in

following table 8.

Table 8 Some important diagnostic species of Cucumber mosaic virus.

Diagnostic species Symptoms

Beta vulgaris Large chlorotic local lesions; not systemic.

Chenopodium amaranticolor and

C. uinoa.

Chlorotic or necrotic local lesions. Rarely systemic.

Cucumis sativus (cucumber) Systemic mosaic and stunting varying in severity with the virus strain.

Cucurbita spp. Systemic mosaic and stunting of varying severity.

Lycopersicon esculentum (tomato) Mosaic and stunting with filiform leaves to different extents, often extreme (fern leaf).

Nicotiana tabacum Mild to severe mosaic and stunting, depending on the virus strain. Some strains induce severe yellow chlorosis. In inoculated leaves, most isolates of Subgroup I (A or B) do not induce symptoms, but most isolates in Subgroup II induce etched rings , as well as in systemically infected leaves in some instances .

Vigna spp. Chlorotic lesions in inoculated leaves of tobacco.

Phaseolus aureus and P. vulgaris cv. Pinto

Small purple necrotic lesions in inoculated leaves.

Vigna unguiculata (cowpea) Large (isolates of Subgroup I A or B) or small (isolates of Subgroup II) brown lesions in inoculated leaves. Only some Subgroup I isolates are systemic causing mild mosaic and these induce chlorotic lesions in inoculated leaves.

Nicotiana benthamiana Severe stunting, leaf curling and leaf deformation, some strains also produce necrosis along veins.

Source: Plant Virus Description (http://www.dpvweb.net/dpv/showdpv.php?dpvno=400).


24

2.3.6. Transmission of CMV

In nature, CMV has been found to be transmitted by several mechanical means, as shown in

the following figure 4.

Transmission of viruses

Nematodes

Insects

Pollens

Mechanical

Grafting

Fungi

Dodder

Fig. 4 Figure show various modes of CMV transmissions in plants occur in nature.

Transmission through vectors/insects

The most notable characteristic of CMV for its natural spread is that it can be transmitted by

numerous species of aphids (Kennedy et al. 1962; Palukaitis et al. 1992; Teixeira da Silva

2003). More than 85 different species, especially Aphis gossypii, Myzus persicae and

Pentalonia nigronervosa, are known to be capable of transmitting CMV (Roossinck et al.

2002). The virus is transmitted from infected plants to healthy ones in a non persistent or

stylet borne manner (Kennedy et al. 1962; Palukaitis et al. 1992). Virus is acquired by the

vector in less than one minute of feeding and there is no latent or waiting period before the

virus can be transmitted. The virus is retained by the aphid for less than 4 h and is not

transmitted to progeny aphids. The efficiency of transmission depends on the host on which

the aphid colonies, the virus source plant, aphid species, test plant, virus strains (Normand

and Pirone 1968; Hamilton 1980) and also depend upon virus coat protein (Gera et al. 1979;

Chen and Francki 1990), 2b suppressor protein (Ziebell et al. 2011).

Transmission through seed

Seed transmission has been reported in more than twenty plant species, with varying

efficiencies from a fraction of 1% up to 50% (Palukaitis et al. 1992). The rate of seed


25

transmission varies in different crops from 0.2% in tomato to as much as 30% among bean

varieties (Marchoux et al. 1975) and therefore is affected by cultivar type, environmental

conditions and CMV strain (Palukaitis et al. 1992). The 10-15% virus transmission trough

seed has also been observed in case of Amaranths in India (Raj et al. 1997a). Virus may be

present in the embryo, endosperm and seminal integuments, as well as in pollen (Yang et al.

1997). Seed transmission in weeds is of epidemiological significance (Quiot et al. 1983; Rist

and Lorbeer 1991).

Mechanical transmission

The most thorough studies on mechanical transmission of CMV were made by Yarwood and

Hecht-Poina (1970), who described their inoculation buffer also. In general, 0.1M phosphate

buffer, pH 7.0 is commonly used for the purpose of sap or mechanical inoculation at a

dilution of 1:10 or 1:20 tissue to buffer (w/v) ratio. CMV is also spread by cuttings taken

from infected perennial woody ornamental species. In case of chrysanthemum, the

propagation through cuttings from an infected source material is a major cause of

CMV/TAV transmission from one to several generations (Raj et al. 2008). At least 10

species of Cuscuta (Dodder) are able to support multiplication and transmit CMV (Francki et

al. 1979).

2.3.7 Serology and relationships of CMV strains

The poor immunogencity of most CMV strains in nature is a major problem in producing

high titre antisera (Francki et al. 1966; Tomlinson et al. 1973) which depends upon route of

administration, dosage, number and frequency of administration and bleeding schedules,

breed and age of rabbit (Ziemiecki and Wood 1975). Immunogenicity may be enhanced by

fixing the virus with formaldehyde (Francki and Habili 1972) or glutaraldehyde (Francki and

Hatta 1980).

Because the virus precipitates on exposure to physiological salt solutions and mild

heating, gel immunodiffusion tests are done usually in buffers of low molarity or water

(Francki et al. 1966; Scott 1968). Polyclonal antibodies show different results depending on

the type of ELISA used: plate-trapped antigen ELISA shows less strain specificity than

double antibody sandwich ELISA , but gives similar overall results to gel immunodiffusion

tests (Wahyuni et al. 1992). Antisera can vary considerably in their ability to detect virus in

ELISA and in immunoblots. Several panels of monoclonal antibodies have been prepared


26

with variation in specificity. The specificity can vary also with the technique used (type of

ELISA or type of immunoblot). Several serological procedures have been used for

characterization of virus from time to time viz. ring precipitin test (Mink et al. 1975), agar

gel immunodiffusion test (Rao et al. 1982), SDS immunodiffusion (Purciful et al. 1981),

Western immunoblot (Hsu et al. 1989), immunosorbent electron microscopy (Francki and

Hatta 1980) and ELISA (Devergne et al. 1981; Rao et al. 1982). Amongst gel

immunodiffusion and ELISA are the two most widely used. Polyclonal and monoclonal

antibodies (MAbs) specific against several strains of CMV have been raised and potentially

used in grouping of CMV strains (Rist and Lorbeer 1989; Ambrosova et al. 1992; Hsu et al.

2000).

Very recently microarrays system was developed to detect and differentiate CMV

serogroups and subgroups (Deyonga et al. 2005). The coat protein genes of 14 new isolates

were amplified using cy3-labelled. These amplicons were hybridized against a set of five

different serotype and subgroup specific 24-mers, bound to an aldehyde-coated glass slide

via an aminolinker.

2.3.8 Biochemical properties

Virions of CMV contain 18.2% nucleic acid; 79.8% protein without any lipid content. Total

genome size is 8.621 kb. Genome divided in three parts; largest genome part is of 3.389 kb;

the 2nd largest 3.035 kb and the 3rd largest 2.197 kb. Genomic nucleic acid was isolated by

Gould and Symons (1977). Base composition of entire genome is about 24% G; 23% A;

23% C; 30% U. 5´terminus of RNA has a methylated nucleotide cap. PolyA region is absent

and genome has t-RNA like activity (Brunt et al. 1996).

2.3.9 Particle morphology of CMV

CMV consists of polyhedral core particles of 29 nm in diameter with T=3 quasi surface

lattice symmetry (Francki et al. 1966; Francki and Hatta 1980) and a hollow centre (Tolin

1977). Lot and Kaper (1976) showed that virions encapsidated either one molecule of RNA1

or one molecule of RNA2 or one molecule each of RNA3 and RNA4. Wikoff et al. (1997)

studied the 3D structure of CMV by cryoelectron microscopy and image reconstruction, and

showed the structural similarity of CMV to Cowpea chlorotic mottle virus (CCMV, family

Bromoviridae).


27

Crystal structure of CMV has been analyzed at 3.20A resolution. Analysis showed that the

subunits form several arrangements with axis of twofold, threefold, fivefold, quasi-threefold

and quasi-sixfold symmetry (Smith et al. 2000). The exterior radius along the quasi-sixfold

axis is 1440A and the density along the fivefold extends 30A more. The RNA is tightly

packed against the protein shell, leaving the hollow core of about 1100A along the threefold

axis. The N-terminal 22 amino acid residues of the capsid protein have a high density of

arginine residue, a net positive charge of +7, and probably interact with the RNA, as well as

this arginine rich residues are essential for the particle formation (Schmitz and Rao 1998). A

unique feature of CMV as compare to other T=3 plant viruses is that N-termini of the B and

C subunits form a hexameric bundle of amphipathic helices that runs parallel to the quasi-

sixfold axes, starting at ~850A from the centre of the virion and extending to the capsid-RNA

interface. One site of the capsid, in an external loop between β strands H and I (the H-I

loop), the sequence of which is conserved among all Cucumoviruses, is involved in metal

interaction (Smith et al. 2000).

Later Pacios and Garcia-Arenal (2006) studied the structure, electrostatic feature and

molecular surface of capsid of CMV which showed that there exists an external region of

negative electrostatic potential that has arisen from strictly conserved charged residues

situated near the external HI loop of the subunits in the capsomers. This negative domain

surrounds the fivefold and quasi-sixfold axes and locates above regions of positive potential

that extend to cover, nearly homogeneously, the inner surface of capsid, where interaction

with encapsidated RNA occurs.

2.3.10 Genome organization of CMV

The multicomponent nature of CMV genome was first discovered by Kaper et al. (1965) by

physico-chemical characterization of a cucumoviruses. Lot et al. (1974) demonstrated that

CMV has a tripartite plus sense RNA genome consisting of four RNA species designated as

RNA1, RNA2, RNA3 and a subgenomic RNA4 responsible for the expression of CP. All the

four RNAs have a 7-methyl guanosine cap at their 5′ ends (Fig. 5) (Symons 1975). For

infectivity, only the three largest RNAs are required (Gould and Symons 1977).

Occasionally a fifth RNA, known as the satellite RNA, about 330-386 nucleotides in

length may be present (Diaz-Ruiz and Kaper 1977; Lot et al. 1977; Gould et al. 1978).

Symons (1979) first published sequences of 270 nucleotide residues from 3′ end of all 4

RNAs of CMV, of which 1-130 initial residues were found to be identical in all RNAs. This


28

region folds into a t-RNA like structure (Rizzo and Palukaitis 1988; Kataoka et al. 1990) and

important for viral RNA replication (Rao et al. 1989).

RNA 1 and 2

The CMV genome has been studied extensively and the replicase functions have been

attributed to RNAs 1 and 2 encoded proteins namely 1a (111 kD) and 2a (94-97 kD)

(Palukaitis et al. 1992; Palukaitis and Garcia-Arenal 2003). Viral RNA replication is

dependent on efficient interaction between these two non-structural proteins encoded by

monocistronic RNAs 1 and 2 (Kao et al. 1992). RNA1 encodes the putative protein with

helicase and has RNA-capping activities (Ahola and Ahlquist 1999; Kong et al. 1999), while

RNA2 encodes the RNA dependent RNA polymerase. Replication of CMV genome requires

the replicase that is composed of a complex of viral encoded 1a and 2a proteins and

unidentified cellular host proteins (Sivakumaran et al. 2000).

Nitta et al. (1988a) showed that tobacco protoplasts infected with RNAs 1 and 2 of

CMV were capable of producing a membrane bound replicase that can synthesize the

replicative forms of CMV RNAs 1 and 2. This indicated that RNAs 1 and 2 encoded proteins

were involved in the formation of viral replication complex like in other tricornaviruses

(Kiberstis et al. 1981).

CMV 2b Protein

The 2b protein, a suppressor of post-transcription gene silencing (PTGS), has been

thoroughly investigated during the past decade (Brigneti et al. 1998; Gonzalez et al. 2010;

Guo and Ding 2002). Previous reports have shown that 2b plays an important role in diverse

processes, including symptom induction as a viral virulence determinant, host-specific virus

accumulation, inhibition of salicylic acid (SA)-induced resistance and systemic spread of

CMV (Brigneti et al. 1998; Ding et al. 1995; Ji and Ding 2001; Li et al. 1999; Shi et al.

2002; Soards et al. 2002). Diaz-Pendon et al. (2007) provided evidence that the subgroup II

strain Q-CMV 2b protein efficiently suppresses 21- and 22-nucleotide viral siRNA-mediated

antiviral silencing by reducing the production of viral siRNA in Arabidopsis. The infection

of tobacco with a 2b gene deletion mutant (CMVD2b) induced strong resistance to aphids

(Myzus persicae) while CMV infection fostered aphid survival (Ziebell et al. 2011).

Moreover, the inhibition of AGO1 by the 2b protein triggers increased expression and

activity of AGO2 (Harvey et al. 2011), but the 2b protein’s ability to inhibit antiviral


29

silencing is most probably mediated through the sequestration of siRNAs rather than the

binding of AGO1 (Gonzalez et al. 2010). Duan et al. (2012) reported that a domain of the 2b

protein from another strain SD-CMV, which is thought to be required for interaction with

AGO1, is spatially separated from the double stranded (ds) RNA binding domain, and

interaction of 2b with AGO1 is dispensable for the suppression of siRNA-mediated RNA

silencing. The CMV-2b protein not only inhibits anti-viral RNA silencing but also quenches

transcriptional responses of plant genes to jasmonic acid (a key signalling molecule in

defense against insects).

5´ cap t RNA 3´

Genomic RNA 1: 3,300 nt

111 K

ORF 1a

5´ cap t RNA 3´

Genomic RNA 2: ~3,057 nt

97 K

ORF 2a ORF 2b

3´ terminal homologuesIntergenic region in RNA 3

5´ terminal homologues

5´ cap t RNA 3´Sub genomic g RNA 4: ~1,000 nt

24 K

ORF 4

CP

t RNA 3´5´ cap

30 KMP

Genomic RNA 3: ~2,200 nt

ORF 3a IR ORF 3b

5´ cap t RNA 3´

13 K

ORF 4a

2b

Sub genomic g RNA 4a: ~700 nt

t RNA 3´RNA 5: ~300 nt

t RNA 3´RNA 3b: ~500 nt, present only in TAV

Fig. 5 Generalized cartoon showing genomic organization of Cucumovirus (family Bromoviridae). Genomes of Cucumber mosaic virus (CMV), Tomato aspermy virus (TAV) and Peanut stunt virus (PSV) each consisted of three genomic RNAs (RNA 1-3) and two major subgenomic RNAs (RNA 4a & 5). CMV and TAV contain a minor RNA (RNA 5), whereas TAV also contains second minor RNA (RNA 3b).


30

This suggested that it might affect interactions between infected plants and aphids,

insects that transmit CMV. 2b protein of CMV-HL strain could bind to Arabidopsis catalase

that is important in scavenging cellular hydrogen peroxide, leading to the induction of

distinct necrosis on Arabidopsis. Very recently, they found that the 2b-AGO1 interaction

affects the biogenesis of trans-acting siRNA by inhibiting AGO1 slicer activity in vivo (Feng

et al. 2013). Self-interaction of the cucumber mosaic virus 2b protein plays a vital role in the

suppression of RNA silencing and the induction of viral symptoms (Xu et al. 2013).

RNA 3 and 4

RNA3 of CMV is di-cistronic and contains two open reading frames (ORFs). The 5′

proximal ORF encodes the 3a movement protein (MP) of 30 kDa whereas the 3′ distal end

encodes the coat protein (CP) about 24 kDa in size. 3a is the translation product of RNA3

but the coat protein is expressed through the subgenomic RNA 4 which is co-linear with the

3′ 1 kb of RNA 3 (Gould and Symons 1978). MP has been identified for a number of plant

viruses and has been found to be responsible for the cell-to-cell movement of the virus in the

host however the mechanism may differ between viruses (Taliansky and Garcia-Arenal

1995). For example, in TMV the MP interacts with plasmodesmata thereby increasing its

permeability facilitating virus movement in non-virion form (Atkins et al. 1991; Wolf et al.

1989), whereas in cowpea mosaic virus, MP induces tubule formation extending from

plasmodesmata of infected cells which are considered to facilitate cell-to-cell movement of

the virus in virion form. Nucleotide sequence of RNA3 from several subgroup I and II

members have been determined (Nitta et al. 1988b; Cuozzo et al. 1988; Quemada et al. 1989;

Owen et al. 1990). A 97-98% homology within subgroup and 74-75% between subgroup I

and II have been found (Palukaitis et al. 1992). When the MP and CP genes were compared

for homology between subgroup I and II and their encoded proteins, it was concluded that

within subgroup I, the percent homology among 3a gene and 3a protein was higher (98-99%

in both) than in CP gene and CP counter parts (96-99%; 94-99%). However, between the

two subgroups, 3a gene shows a homology between 78-99% and its protein of 80-84%. On

the other hand, CP shows a homology of 80-83% whereas CP gene of 76-77% (Palukaitis et

al. 1992).

The coat protein (CP): determinant of symptom, host range and aphid transmission

CMV capsid consists of 180 identical protein subunits (Finch et al. 1967) of about 24k Da

(Habili and Francki 1974). The capsid has an external and internal diameter of 29 nm and


31

16.5 nm respectively (Jacrot et al. 1977). The subunits of CP are arranged in pentamer-

hexamer clusters with T=3 icosahedral surface lattice symmetry (Smith 2000) and has

central core of each hexamer and pentamer (Wikoff et al. 1997). CP usually consists of 218

amino acid residues in all CMV strains as revealed by electrophoretic mobility data

(Palukaitis et al. 1992). CPs are named for their primary function; to encapsidate viral

genomic nucleic acids. However, encapsidation is only one feature of an extremely diverse

array of structural, functional, and ecological roles played during viral infection and spread

in nature or through aphids (Callaway et al. 2001). The known roles of CP established

besides encapsidation are systemic movement (Suzuki et al. 1991), host range (Shintaku and

Palukaitis 1990) and aphid transmission (Gera et al. 1979, Perry et al. 1994; 1998).

Shintaku et al. (1992) delineate that the symptoms expression, chlorosis phenotype, is

associated with a particular local secondary structure surrounding amino acid 129 in the CP,

on the basis of their study on two strains of M-CMV (a chlorotic strain) and Fny-CMV (a

green mosaic strain). They demonstrated that the change in the codon for leucine at position

129 of CP in M-CMV to proline 129 of Fny-CMV changed the phenotype from chlorotic to

green mosaic whereas the opposite phenotype was observed when proline 129 in Fny-CMV

CP was altered to serine 129. Likewise Szilassy et al. (1999) showed that stunting induced in

infected N. glutinosa was determined by single amino acid lysine at position 193 in CP.

CP is also a primary determinant of aphid transmission in CMV and a number of

non-persistently transmitted plant viruses (Gera et al. 1979; Pirone and Blanc 1996). Smith

et al. (2000) studied of CMV at 3.2ºA resolution using X-ray crystallography and

demonstrated that the residues in CP important for aphid transmissibility lie at the outermost

portion of the H-I loops. The work also yields details of the portions of the virus that are

hypothesized to mediate binding to aphid mouthparts. Liu et al. (2002) observe that changes

in CP could influence transmission and transmission phenotype depending on transmitting

aphid species.

CP as characterizing protein

Several strains of CMV have been identified in the past few decades on the basis of physical

and chemical properties (Francki et al. 1966). High similarity in the properties of CMV to

those of PSV (Boatman et al. 1973) and TAV (Habili and Francki 1974) supported the

grouping of these viruses as cucumoviruses (Harrison et al. 1971). Computer assisted

sequence comparisons of the amino acid sequences of the CP gene of various CMV strains


32

suggested that CP of CMV strains that belonged to same subgroup were strongly conserved

(Owen et al. 1990, Deyong 2005).

Edwards and Gonsalves (1983) proposed division of CMV strains into two subgroups

based on peptide mapping of the viral CPs for the first time. In order to determine the extent

of genetic variation between the strains and to map the genetic variation Winter et al. (1985)

used the RNA protection assay to analyze RNA3 of 12 CMV strains, supported the

relationships proposed by (Palukaitis et al. 1992). This was consistent with the divisions of

CMV proposed on the basis of symptomatology and serological properties proposed by

Owen and Palukaitis (1988). However, strains within a subgroup could not be further

differentiated by these techniques.

A major breakthrough occurred when Palukaitis and Zaitlin (1997) on the basis of

nucleotide sequence analysis of CMV strains proposed splitting of subgroup I into IA and

IB. The Asian strains were placed under the subgroup IB while all the other strains

originating from Australia, Japan, Europe and North America were referred to as subgroup

IA. They proposed the splitting based on the observation that all the Asian strains differed

from other subgroup I strains by 7-10% whereas they differed only by 2-3 % from each other

(Sialer et al. 1999). Roossinck et al. (1999) indicated radial evolution of three subgroups

based on the studies of rearrangements in the 5′ non-translated region and phylogenetic

analysis of CMV RNA 3. Alignment of 5′ UTR of RNA 3 of 26 strains of CMV suggested

division of CMV into IA, IB and II subgroups with IA and IB arising from the further

division of subgroup I.

In concurrent scenario, Roossinck (2002) did complete phylogenetic analysis of 15

CMV strains whose complete nucleotide sequences had been determined. The trees

estimated for ORFs located on the different RNAs were not found to be congruent and did

not completely support the sub grouping indicated by the CP ORF, indicating that different

RNAs had independent evolutionary histories. It was suggested that reassortment has an

important role in the evolution of the virus. The evolutionary trees of the 1a and 3a ORFs

analyzed were also found to be more compact and displayed more branching than did those

of the 2a and CP ORFs which may reflect more rigid host-interactive constraints exerted on

the 1a and 3a ORFs. In addition, analysis of the 3′ NTR that is conserved among all RNAs

indicated that evolutionary constraints on this region are specific to the RNA component

rather than the virus isolate. This indicates that functions other than replication are encoded

in the 3′ NTR. The analysis proposed that reassortment leading to the genetic diversity found


33

among CMV strains could be a contributory factor in the enormous evolutionary success of

CMV (Roossinck 2002; Palukaitis and Garcia-Arenal 2003).

RNAs 4a, 5 and 6

CMV strains belonging to subgroup II (Q, Kin and S) contain minor RNAs (4a, 5 and 6)

(Peden and Symons 1973). RNA5 is co-terminal with the 3′ 304 nucleotides of RNAs 3 and

4 whereas RNA 4a is approximately 680 nucleotides co-terminal with 3΄ end of RNA2 and

encodes the 2b protein (11.3kDa) that is expressed in vivo (Ding et al. 1994). The role of this

protein has been demonstrated in systemic infection, pathogenicity and suppressing post-

transcriptional gene silencing (PTGS) (Brigneti et al. 1998; Mayer et al. 2000; Ji and Ding

2001; Guo and Ding 2002; Cillo et al. 2002). CMV strains of both subgroups also contain

low (1-5%) RNA6 that comigrates on polyacrylamide gel, yet its origin and requirement for

CMV is still unknown.

Satellite RNA/CARNA5

Some strains of CMV contain a 340 to 400 nucleotides single stranded small RNA parasite

molecule ca. Satellite RNA (satRNA), a type of noncoding RNA which is dependent on

CMV (a helper virus) for its multiplication in the infected plant and has regulatory effect on

the expression of disease symptoms but is not required for replication of the virus (Rossinck

2001). To date, over 100 CMV satRNA variants have been found to be associated with over

65 CMV isolates (Palukaitis and Garcia-Arenal 2003). Kaper et al. (1993) identified and

characterized a 334 nucleotide necrogenic CARNA-5, isolated from tomato fields in

southern Italy, cause of massive outbreak of lethal necrosis in the summer of 1988. It

provided the first direct evidence of the involvement of a viral satellite in epidemics. A new

satellite RNA is associated with natural infections of cucumber mosaic virus in succulent

snap bean were reported from Wisconsin (Nouri et al. 2012)

Mostly, presence of sat-RNA result in decreased accumulation of CMV in the tissues

of infected plants (Waterworth et al. 1979) and modifies the symptoms induced by CMV as a

result of a complex interaction between the strain of CMV, the variant of satellite RNA and

the species of host plan. Raj et al. (1999a) also demonstrated the role of satellite RNA of an

Indian isolate of CMV in inducing lethal necrosis of N. benthamiana plants. They showed

that the RNA preparations without satellite RNA did not induce symptoms as severe on N.


34

benthamiana as those with satellite RNA. Satellite RNA reduces expression of the 2b

suppressor protein resulting in the attenuation of symptoms caused by CMV infection.

Mechanisms of sat RNA-mediated symptom modification

Three distinct mechanisms of sat RNA-mediated symptom modification have been

reported.D strain sat RNA (D-sat)-induced lethal systemic necrosis in tomato plants,

occurring in Europe, has been demonstrated by Xu and Roossinck (2000). D-sat causes

nuclear DNA fragmentation and chromatin condensation in necrotic tissues, implicating

programmed cell death in D-sat-induced necrosis. In addition, the authors found that the

spatial patterns of necrosis induction, vascular cell development and D-sat localization in

tissues were correlated. D-sat may alter normal vascular cell development and lead to

programmed cell death, probably via xylogenesis or senescence.

Y strain sat RNA (Y-sat), one of several well-known sat RNAs, causes bright

yellowing in some solanaceous species, including N. benthamiana, tobacco and pepper.

Recently, striking reports on the mechanism of Y-sat-induced chlorosis have been published

separately by Shimura et al. (2011). Y-sat includes a 22-nucleotide homology sequence that

is complementary to the tobacco magnesium protoporphyrin chelatase subunit I gene (ChlI,

key enzyme for chlorophyll synthesis), and the amount of ChlI mRNA is reduced in the

chlorotic region of Y-sat infected tissues. Tobacco plants in which the expression of ChlI

mRNA was expressed by an RNAi construct or by CMV vector showed chlorotic

phenotypes similar to transfection with Y-sat. Interestingly, Y-sat could not induce chlorosis

in plants encoding a

2.3.11 Identification, characterization/classification of CMV strains

Strains of CMV have been classified on the basis of serological typing, peptide mapping of

the coat protein (Edwards and Gonsalves 1983) and sequence similarity of their genomic

RNAs (Gonda and Symons 1978, Owen and Palukaitis 1988). These classifications divided

CMV strains into either Subgroup I or Subgroup II (Owen and Palukaitis 1988). Overall

nucleotide sequence similarity among isolates is 90% to 98% within Subgroup I and about

98% within Subgroup II; similarities between isolates from different subgroups are 65% to

70%. Analysis of RNA 3 sequences of 26 isolates indicates that Subgroup I strains can be

subdivided further into Subgroups IA and IB, in which Subgroup IA strains are more closely

clustered than are Subgroup IB strains. Subgroups IA and II appear to be monophyletic


35

(Roossinck et al. 1999). Similar relationships are obtained comparing sequences of RNAs 1

or 2 (Roossinck 2002). Cross protection occurs between strains from all subgroups. CMV is

related to Tomato aspermy virus) and to Peanut stunt virus (PSV, Mink 1972) the two other

definite members of the genus Cucumovirus; Robinia mosaic virus is now considered a

strain of PSV. Nucleotide sequence similarity amongst these three cucumoviruses is about

60% to 65% and they can be differentiated serologically and by host range (Kaper and

Waterworth 1981).

Monoclonal antibodies have been obtained that can distinguish CMV Subgroup I and

Subgroup II isolates, and/or react with most of the isolates (Wahyuni et al. 1992). RT-PCR

has been used to detect cucumoviruses and differentiate isolates in the CMV subgroups

(Choi et al. 1999). Pseudo-recombinant viruses that multiply efficiently can be obtained by

exchange of genomic segments between CMV isolates from both subgroups, and to a lesser

extent, between CMV and the other cucumoviruses (Habili and Francki 1974a; Marchoux et

al. 1975; Rao and Francki 1981;1982). Pseudo-recombinants are also isolated by aphid

transmission from mixed infections (Perry and Francki 1992; Fraile et al. 1997).

Nevertheless, pseudo-recombinants between Subgroups I and II have not been reported in

nature, and those produced between strains in the same subgroup are infrequent (Fraile et al.

1997). A natural pseudo-recombinant between CMV and PSV has been reported.

Mireille Jacquemond in 2012 reviewed CMV in details. This review focuses on those

areas where most progress has been made over the past decade in our understanding of

CMV. Clearly, a deep understanding of the role of the recently described CMV 2b gene in

suppression of host RNA silencing and viral virulence is the most important discovery.

These findings have had an impact well beyond the virus itself, as the 2b gene is an

important tool in the studies of eukaryotic gene regulation. Protein 2b was shown to be

involved in most of the steps of the virus cycle and to interfere with several basal host

defenses. Progress has also been made concerning the mechanisms of virus replication and

movement. However, only a few host proteins that interact with viral proteins have been

identified, making this an area of research where major efforts are still needed. Another area

where major advances have been made is CMV population genetics, where contrasting

results were obtained. On the one hand, CMV was shown to be prone to recombination and

to show high genetic diversity based on sequence data of different isolates. On the other

hand, populations did not exhibit high genetic variability either within plants, or even in a

field and the nearby wild plants. The situation was partially clarified with the finding that


36

severe bottlenecks occur during both virus movement within a plant and transmission

between plants. Finally, novel studies were undertaken to elucidate mechanisms leading to

selection in virus population, according to the host or its environment, opening a new

research area in plant–virus coevolution.

2.4 Management strategies of Cucumber mosaic virus

Cucumber mosaic virus cause significant losses to most of the major crops, around the

world, therefore are the bottlenecks to the crop production (Hull and Davies 1992; Valkonen

1998; Raj et al. 2008b). Management of viral diseases is much more difficult than that

of diseases caused by other pathogens (Verma et. al. 2002) because of the viral diseases

have a complex disease cycle, efficient vector transmission and no effective virtcides.

Integration of various approaches like the avoidance of sources of infection, control of

vectors, cultural practices (conventional) and use of resistant host plants (non conventional)

have been used for the management of viral diseases of plants.

2.4.1 Conventional measures

Sanitation of cropping filed and planting practices

Cleaning and eradication of infected plant material together with the potential reservoir of

cucumovirus from the fields was found to be very effective method. Perennial weeds should

be eradicated from around greenhouse, gardens and fields to eliminate possible source of

CMV (Agrios 1978; Raj et al. 2008b). Along with this, the practices such as early plantation,

plant spacing use of silver or white coloured mulches were found effective in reducing

disease incidence and obtaining maximum yield . In one study, observed that supplemental

blue-light in the greenhouse enhances Orius oncidiosus, a natural predator of western flower

thrips, reproduction, especially in biological control programmes.

Biological control of virus vectors

Biological control of aphids (the virus vector) capable of transmitting various type of

viruses, especially the cucumo- (CMV and TAV) and potyviruses (BYMV) in several plant

species has been attempted. The feeding behavior of lady bird (Coceinella transversalis)

predator of green and black aphids has been observed on chrysanthemum plants. Different

larval stages as well as adult ladybird predator have been exploited for minimizing the aphid

population (Raj et al. 2005a).


37

Spray of insecticide

Application of insecticides viz. Malathian, Rogor (0.1%) solution either by spraying or

drenching soil, will effectively reduced the virus burden on commercial crops . The regular

use of insecticide should not be in practice because it adversely effects the environment,

round other way, may diminish the quality of crops. Three sprayings of Malathian

insecticide (0.2%) at every 21 days intervals were found to be effective for management and

minimizing the population of both the insect vector borne diseases. An improvement in

growth and yield performance of treated plants was noticed as compared to the non-treated

plants.

Use of anti-viral agents

Inhibitors against virus infection function by stimulating the in-built resistance of susceptible

hosts by both at treated site and at a distance from the primary treated site, a condition

referred to as systemic induced resistance (SIR) (Srivastava 1999). Various plants showing

promising antiviral systemic resistance inducing properties have been reported like

Boerhaavia diffusa, Clerodendron aculeatum, Bougainvillea spectabilis etc (Verma 1985;

Verma and Prasad 1992; Verma et al. 1995; 1998). Other inhibitors of virus infection apart

from plant extracts are micro-organisms and insects, oxidized phenolic compounds,

ribonucleases, dyes and milk (Mehrotra 1991).

Use of virus-free planting material

Viruses spread from mother plant to their progenies by infected cuttings, tubers and other

plant vegetative plant materials have great possibility of virus transmission (Gera and Zeidan

2006; Wang and Valkonen 2008). Use of virus-free planting material and their

transplantation in greenhouses that isolates crop from other plants which harbour or may

harbour viral diseases e.g. susceptible crops, should be practiced for better crop production

yield (Agrios 1978). Using planting material from which all infected plants have been

rogued, applying heat therapy (35-54ºC), use of meristem tip cultures, cold treatment and

chemotherapy are other means suggested for obtaining virus free plants (Raychaudhuri and

Verma 1977). Most strains of the virus can be eliminated from chrysanthemum after 4 weeks

at 37°C; or by meristem-tip culture. Morel and Martin (1952) first demonstrated the

elimination of viruses from dahlia using meristem culture.


38

Since then, the use of meristem culture to obtain virus-free ornamental plants has

been widely used by numerous groups of researchers (Table 9).

Table 9 Elimination of viruses from ornamental plants by shoot meristem culture or its

combination with other biotechnological procedures.

Species Procedure Virus Reference

Alstromeria sp. meristem culture Alstroemeria mosaic virus (AlMV) Chiari and Bridgen 2002

Chrysanthemum sp. meristem culture Cucumber mosaic virus (CMV) Verma et al. 2004b

Chrysanthemum morifolium

cv. Regol Time

meristem culture,

chemotherapy and

thermotherapy

Chrysanthemum B Carla virus (CVB) Ram et al. 2005

Chrysanthemum morifolium

meristem culture mixed infection by CMV and Tomato aspermy virus (TAV)

Kumar et al. 2009

Chrysanthemum sp.

meristem culture Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV),Iris yellow spot virus (IYSV)

Balukiewicz and

Kryczynski 2005

Dianthus gratianopolotanus

meristem culture Carnation mottle virus (CarMV),Carnation latent virus (CLV), potyviruses

Fraga et al. 2004a

Lilium sp. meristem culture Lily symptomless virus (LSV) Allen 1975

L. x elegans

meristem culture and thermotherapy

LSV Nesi et al. 2009

New Guinea Impatiens

(I. hawkerii)

meristem culture Mixed infection by TSWV and CMV Gera and Dehan 1992

I. hawkerii meristem culture TSWV Milošević et al. 2011

Phlox paniculata meristem culture and thermotherapy

CLV, CarMV, CMV, Tobacco mosaic virus (TMV), Tospoviruses (subgroups I, II and III), Potyviruses

Fraga et al. 2004b

Viola odorata

meristem culture Viola mottle virus (VMV), CMV, Bean yellow mosaic virus (BYMV)

Van Caneghem et al. 1997


39

2.4.2 Non-conventional measures

All these approaches are important, but most practical approach is the use of varieties, which

resist vectors, seed transmission, symptom development, cell-to-cell movement and virus

multiplication. Genetic engineering brings new hope for overcoming various

drawbacks associated with conventional breeding for developing crop varieties with

durable resistance by 'pyramiding' genetically engineered resistance over intrinsic plant

resistance. This approach has been successfully applied to generate virus resistant

transgenic plants (VRTPs). The major breakthrough start with the contribution of Powell-

Abel et al. (1986) who genetically engineered virus resistance in tobacco plants using coat

protein (CP) gene from TMV in tobacco using Agrobacterium tumefaciens and the resultant

transgenic plants were confirmed for the presence of foreign DNA sequence and nptII gene

neomycin phosphotransferase), in both primary transformants and their progeny, which

showed virus resistance. Till then, engineered resistance to viruses has been achieved in

various plants either by the use of viral genes (known as the viral derived resistance) or

through the expression of non-viral genes from (b) Plants, and (c) other sources (Table 10).

Table 10 Possible transgenic interference with major events during plant-virus-vector infractions.

Source: Varma et al. 2002.

Trans-gene Transgenic Interference Virus-derived Coat protein Transmission, Uncoating, Assembly Replicase protein Replication Movement protein Invasion Viral protease Protein processing Helper protein Delays symptom development Seed transmission factor Seed transmission Non coding region Competition for viral replicas Antisense RNA/DNA Replication, translation, assembly Ribozyme Replication, translation, assembly Satellite Replication Defective interfering RNAs Replication Plant-Derived Antiviral protein Multiplication Host ‘R’ gene Multiplication Others Plantibodies Replication, protein processing, assembly Yeast RNase Cleaving of dsRNA


40

Virus-derived Resistance (VDR)/Pathogen Derived Resistance (PDR)

The concept that the viral genes, either as a wild type or mutant, could confer resistance in

host plants (Hamilton 1980; Sanford and Johnston 1985), stimulated research for

generating VRTPs through genetic engineering, The first VRTP using VDR was produced

in the mid 1980s by expressing TMV was produced in the mid 1980s by expressing TMV

coat protein gene in transgenic tobacco plants (Powell-Aabel et al. 1986). Since then, many

different viral genes and viral associated RNAs have been used as transgenes to confer

resistance in plants, and VDR became a reality against a range of plant viruses having

positive-sense ssRNA, ambisense RNA or ssDNA (Grumet 1994). This approach has also

been used to develop resistance to viroids (Atkins et al. 1995).

Viruses depend on the host machine for replication. The genome of plant viruses is

ssDNA, dsDNA, ssRNA or dsRNA. Most of the plant viruses have positive sense ssRNA

genome that replicate by virus encoded RNA dependent RNA polymerase and form dsRNA

replicative intermediate. The viral genome is encapsidated in particles having icosahedral

or spiral symmetry formed by compact arrangement of coat protein subunits in specific

pattern. The viral genome could be either monopartite ('undivided) or bi or multipartite

(divided into two or more molecules). Irrespective of the number of genomic molecules,

each genome has open reading frames (ORFs) to produce structural and nonstructural

proteins for various functions l ik e replication, cell-to-cell movement, vector

transmission, encapsulation, etc. The events that follow Infection include disassembly of

virus particles, synthesis or transcription of mRNA (where required), translation of proteins

coded by viral genome for various functions, maturation of particles, systemic

infection and vector transmission. Strategies directed to interference with any of these

functions result in the development of VRTPs (Table 11). Both the coding and non-

coding regions of viral genomes have been used for developing VRTPs. For some groups of

viruses, like potyviruses, resistance in plants has been obtained using almost all the viral

genes as transgenes. CP gene is the most commonly used transgene for developing VRTPs

against viruses belonging to different groups followed by the replicase protein and

movement protein genes (Table 11).

Coat Protein mediated resistance (CPMR)

CPMR has been described as the resistance caused by the expression of a virus capsid/coat

protein (CP) gene in transgenic plants (Beachy et al. 1990). The CP gene is the most widely


41

and commonly used transgene for which virus resistant transgenic plants have been

developed, followed by replicase and the movement protein genes. CPMR has been found

effective irrespective of virus particle morphology (rigid rod, flexuous, isometric,

bacilli form, lipoprotein enveloped), genome organization (positive-sense, negative-

sense, ambisense; monopartite, multipartite) and mode of transmission (mechanical, seed,

pollen, and vector). (Varma et al. 2002). CP-mediated protection has been found to confer

resistance against infection by the homologous virus (depends on the similarity between the

amino acid sequence of the CP) and shown to provide little or no protection against

unrelated viruses (Lomonossoff 1995; Nakajima et al. 1993). However, there were instances

where the resistance spectrum conferred by a coat protein gene was quite broad, as showed

for cucumoviruses and potyviruses (Stark and Beachy 1989; Namba et al. 1991; Nakajima et

al. 1993; Srivastava et al. 2008). Namba et al. (1991) found that the coat protein CMV-WL

provides protection against several subgroup I strains.

Pathogen-derived resistance has been observed to be mediated either by the protein

encoded by the transgene (protein-mediated) or by the transcript produced from the

transgene (RNA-mediated) also known as post transcriptional gene silencing (PTGS) or both

(Varma et al. 2002). When a single copy of CP transgene is inserted, induced protein-

mediated resistance, undergoes transcription and translation resulting in high protein

expression level (Varma et al. 2002). Resistance so expressed is of moderate level and

influenced by the level transgene expression. The in vivo plant expressed CP interfere with

the un-coating of virions, inhibiting both the establishment of infections and the spread of

virus from cell to cell (Wisniewski et al. 1990; Beachy et al. 1990: Srivastava et al. 2008).

CP gene induced resistance is termed as RNA-mediated when multiple copies of

transgene are inserted (Lomonossoff 1995). Resistance so expressed has been observed to be

of high level and strain specific and attributed to lower levels of transcripts. Transgene

expression should be up to mRNA level with little or no transgenic protein. When mRNA

accumulation exceeds a threshold level, co-suppression (gene silencing) is initiated, affecting

transgene expression and virus multiplication (Varma et al. 2002) in a sequence specific

manner (English et al. 1996). The defense system exhibited by such plants results in the

degradation of mRNA produced both by the transgene and the infecting virus (Waterhouse et

al. 2001). This virus resistance mechanism was referred to as homology-dependent virus

resistance to reflect the relationship with homology dependent gene silencing .The

phenomenon has been termed as the post-transcriptional gene silencing or PTGS


42

(Baulcombe 1996). In one case of CMV, whole plants were protected against systemic

infection by both virions and RNA but protoplasts were protected against virions only and

not RNA (Okuno et al. 1993). These observations suggested that more than one mechanism

might be operating, one at the cellular level likely to involve interference with un-coating

and another at cell to cell or whole plant level (Grumet 1994; Kalantidis et al. 2002).

The application of CPMR against CMV was first demonstrated in tobacco (Cuozzo

et al. 1988) and has been applied effectively to mainly vegetable crops, such as tomato

(Fuches et al. 1996; Gielen et al. 1996; Kaniewski et al. 1999; Xue et al. 1994;), cucumber

(Gonsalves et al. 1992), squash (Tricoli et al. 1995), and melon (Gonsalves et al. 1994)

using the CP genes of various CMV strains. Raj and co-workers (2005b) also regenerated

virus-resistant transgenic tomato plant expressing CP gene of Tomato leaf curl virus (TLCV)

using pROK2 expression vector transformed in A. tumefaciens. T0-generation putative

transgenic plants obtained were screened by PCR, Southern and Northern hybridization tests

and Western blot assay, which confirmed the incorporation and expression of the CP gene.

T1-generation transgenic showed significant degrees of disease resistance/tolerance

compared to the untransformed control.

An attempt was also made in India to provide the dual virus resistance (against CMV

and ToLCV) by Praveen et al. (2006). CMV-CP and ToLCV-Rep were transcriptionally

fused under the control of CaMV 35S promoter and tobacco and tomato explants were

transformed using Agrobacterium. They show transforming ToLCV and CMV infected

plants with the homologous chimeric gene construct that produces RNAs, capable of duplex

formation, confers gene silencing and proposed that the antisense suppression in ToLCV

infected plants provides a threshold level of dsRNA needed to induce gene silencing whereas

sense suppression in CMV infected plants may be operating through co-suppression, leading

to delayed and attenuated symptoms (Praveen et al. 2006).

Thereafter, CP-mediated resistance against an Indian isolate of the CMV of subgroup

IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium

tumefaciens-mediated transformation (Srivastava and Raj 2008). Transgenic lines exhibiting

complete resistance remained symptomless throughout life and showed reduced or no virus

accumulation in their systemic leaves after virus challenge. These lines also showed virus

resistance against two closely related strains of CMV (Srivastava and Raj 2008). Raj and co-

workers used CPMR strategy in chrysanthemum (Kumar et al. 2012) and tomato (Pratap et

al. 2012b) also for development of virus resistant transgenic plants against CMV.


44

Replicase protein

Replicase protein gene is second widely used transgene to confirm resistance against plant

viruses and the strategy is referred to as replicase protein-mediated resistance (RPMR). RPMR

gives nearly immune type and highly specific resistance for the virus from which the transgene

is isolated. It more effective than CPMR and is not influenced by inoculums levels.

Movement protein

In this strategy defective movement proteins have been expressed in transgenic plants. In

contrast to other strategy, this approach offers attractive possibility to confer broad spectrum

resistance to related and unrelated virus. For example, a defective TMV MP expressed in

transgenic tobacco plants was shown to confer varying levels of resistance to a number of

viruses that are not members of tobamovirus group, including AMV, CMV, PCSV, TRV, and

TRSV.

Satellite and defecting interfering RNAs

Some viruses have specific satellite RNA molecules (sat-RNA), which is considered virus

parasites being dependent on helper virus for multiplication. The sat-RNAs of the latter type

have been used for developing VRTPs for resistance to cucumo- and nepoviruses. Transgenic

tobacco plants expressing sat RNAs of CMV or TRSV on challenge inoculation exhibited

attenuation of disease symptoms (Harrison et al.1987).

Research efforts for developing viral resistant transgenic plant in India

Certain groups of plants virus like gemini, poty, cucumo, badna-, and tobamoviruses are major

constraints of crop production in the Indian subcontinent. Beside these, tospo- and ilarviruses

are emerging as threatening pathogens. Development of host plants resistance for the

management of viral diseases is the most practical approach used in the country, but for a large

number of host-virus combinations either suitable sources of resistance are not available or the

resistance genes are linked to undesirable agronomic traits. It is therefore essential to launch an

aggressive program for developing VRTPs. Efforts are in progress at various centers in the

country to develop VRTPs of cotton, mung bean, papaya, potato, tomato and soybean resistant

to the important viruses affecting these crops (Table 12).


45

Table 12 Research efforts for developing viral resistant transgenic plant in India (Varma et al.

2002).

Crop Transgene Resistant against Research centre

Cotton Replicase (CuCLV) Cotton leaf curl disease IISc, Bangalore

Mungbean CP/replicase (MYMV) Mungbean yellow mosaic

Madurai University

Papaya CP (PRSV) Papaya ringspot IARI, New Delhi

Potato CP (PVY) Potato virus Y IARI, New Delhi; CPRI, Shimla; BARC, Mumbai

Rice CP (RTSV/RTBV) Rice Tungro South campus, Delhi University

Soybean Replicase (MYMV) Soybean yellow mosaic IARI, New Delhi

Tobacco CP (PVY) Patato virus Y IARI, New Delhi

Tomato Replicase (ToLCV), CP (CMV). CP (ToMV)

Tomato leaf curl, tomato mosaic

IARI, New Delhi NBRI, Lucknow

Chrysanthemum CP (CMV) Chrysanthemum mosaic NBRI, Lucknow

Status of the application of pathogen-derived resistance against virus of ornamental crops

The current technical limitation for generating transgenic virus resistant floral crops is not so

much the viral resistance strategy but rather the gene delivery (transformation) system that must

be developed for each crop. To date, by using Agrobacterium-mediated or by microprojectile

bombardment gene-delivery methods, successful transgenic ornamentals have been obtained for:

alstroemeria, chrysanthemum , carnation, gerbera, lisianthus, rose, alstroemeria , lily, tulip and

gladiolus, Nevertheless, except transgenic chrysanthemum plants expressing N gene of TSWV,

In India, Kumar and co-workers developed virus resistance chrysanthemum plant against CMV,

using agrobacterium transformation harboring pROK2 vector chimeric with CMV-CP gene.

Transformation efficiency of gene was ~6% and 12.5 % transgenic chrysanthemum plants were

showed delay symptoms compared to non transgenic plant (Kumar et al. 2012).

2.4.3 Genetic transformation in ornamental plants

Since floricultural and ornamental crops are grown for aesthetic purpose and are non-edible

there is likely to be less concern in bio safety issues compared to other food crops. Hence there


46

is considerable potential for developing transgenics in ornamental crops. Advances in transgenic

technology provide new opportunities for manipulation of the genome. These will have

significant impact on expanding and diversifying the gene pool of crop plants, introducing

specific genes and shortening the time required for the production of new varieties or hybrids.

Genetic transformation is beneficial to increase the production and quality by creating

plants with enhanced resistance to diseases, insects or viruses and increased tolernce to

environmental stresses like salinity, temperature or drought. Through this technique genes for

shelf life, flower color and architecture may be directly transferred to develop new varieties that

are tailor made to customer preferences. So, there are an immense potential in the improvement

of ornamentals through transgenic technologies.

The gene transfer method to be used must be compatible with the plant genotype and the

tissue to be treated. In ornamental plant Genetic transformation could be perform by Electro

injection, Microparticle bombardment, Seed inbibitions, Agrobacterium-mediated and Pollen

tube mediated DNA delivery methods, among these Agroacterium –mediated transformation is

very efficient, natural and most common method.

Table 13 Deferent mode of gene transformation method in ornamental plants. Gene Transformation Method Explants References 1. A. tumefaciens mediated chrysanthemums, gerbera, rose,

sedum, Oncidium Rhododendron, carnation phalaenopsis Arabis sp Anthurium, Phalaenopsis Artemisia endrobium,Agapanthus

Swarnapiria 2009

2. Microparticle bombardment Orchid Cymbidiums, gladiolus Cv Jenny Lee Brassia, Cattleya and Doritaenopsis, D. nobile

Men et al. 2003 Knapp et al. 2000

3. Seed imbibitions Orchid Chia et al. 1994

4. Pollen tube mediated DNA delivery

Orchid

Nan and Kuehnle 1995

5. Electroinjection Orchid Yu et al. 1999


47

Agrobacterium mediated transformation: The naturally evolved unique ability of A.

tumefaciens to precisely transfer defined DNA sequences to plant cells has been very effectively

utilized in the design of a range of Ti plasmid-based vectors (Gelvin 2000; Zupan et al. 2000).

Three genetic elements, Agrobacterium chromosomal virulence genes (chv), T-DNA delimited

by a right border and a left border and Ti plasmid virulence genes (vir) constitute the T-DNA

transfer machinery.

The mechanisms governing the transfer of ‘T-complex’ via the conjugation channel and

the roles of plant and Agrobacterium proteins in T-DNA integration are being intensely studied.

Agrobacterium-based DNA transfer system offers many unique advantages in plant

transformation: (1) the simplicity of Agrobacterium gene transfer makes it a easy vector, (2) a

precise transfer and integration of DNA sequences with defined ends, (3) a linked transfer of

genes of interest along with the transformation marker, (4) higher frequency of stable

transformation with many single copy insertions, (5) reasonably low incidence of transgene

silencing and (6) the ability to transfer long stretches of T-DNA.

Key factors affecting stable transformation of Agrobacterium-transformation of

ornamentals: Following factors are seriously affected regeneration and transformation

efficiency of ornamental plants:

Explants for genetic transformation: Selection of suitable explants is primary need for

transformation because regeneration, bacterial infection and selection of particular transform

cell completely depend upon explants.

Bacterial density: Concentration of bacterial cells in the induction medium is another important

factor to be considered for efficient transformation. Very low density of bacterial population

could lead to ineffective transformation, whereas very high density may lead to necrosis and

death of the explants. Some species are very sensitive to bacterial infection and hence very low

density of bacterial population is used. Bacterial conc. OD = 0.1-0.6, at 600nm wavelength are

generally used to infection in ornamental for successful gene transformation (Swarnapiria

2009).

Co-cultivation: The explant chosen, in its most receptive stage, is exposed to the

Agrobacterium culture in the induction media at an optimum bacterial density. Both the


48

composition of the induction media and the time of induction play a key role in the efficiency of

transformation.

Addition of phenolic compounds: Agrobacterium tumefaciens respond to certain phenolic

compounds such as acetosyringone and hydroxyacetosyringone which are excreted by wounded

plants. These small molecules act to induce the activity of virulence (vir) genes that are encoded

on the plasmid.

The ‘vir’ genes are located on 35Kb region of the plasmid that lies outside the TDNA

region. When A. tumefaciens get attached to a plant cell, and the ‘vir’ genes are induced, then

‘T-DNA’ (which contains the gene of interest) is transferred to plant cell. The transformation

frequency was improved by the presence of acetosyringone (100 mM) in co-cultivation medium.

The effectiveness of acetosyringone might be assumed due to the vir G gene harboured by

pTOK233 was activated by the presence of acetosyringone (Mishiba et al. 2000).

Elimination of bacteria after co-cultivation: Complete elimination of bacteria from the

explant after co-cultivation is very essential; otherwise it will interfere with the growth and

organogenesis of the explant. Overgrowth of bacteria causes death of the explant and disrupts

the experiment. Elimination of bacteria from the explant is done by the use of antibiotics. The

antibiotic chosen should be such that it efficiently kills the bacteria at the same time it does not

affect the growth and organogenesis of the explants.

The most commonly used antibiotics for this purpose are carbenicillin and cefotaxime.

However, their effect on the explant has to be studied before choosing any one of them as they

are also reported to have detrimental effect on some species. Literater reported worker usewide

range of cefotaxime concentration warier from 50-800 mg/l. but most of worker used

cefotaxime conc. between 200-400 mg/l (Swarnapiria 2009)

Selection of Transformed Cells: Screening of untransformed cells or selection of transformed

cells is an important aspect of transformation work. The selection agent must be toxic to un-

transform plant cells. Thus the most effective toxins are those which either inhibit growth of

untransformed cells or slowly kill the untransformed cells. Optimal selection pressure will use

the lowest level of toxin needed to kill the untransformed tissues. Different workers are used


49

wide range of kanamycin concentrations from 7 to 200 mg/l (Swarnapiria 2009) in deferent

plant and varieties.

Agrobacterium - transformation of petunia for developing virus resistance plant

Kim and co-author (1995) was transformation CMV I17N satellite RNA of petunia plants using

Agrobacterium. Leaf explants was used for co-cultivation with A. tumefactions (LBA4404)

harboring plasmid pROK1/105. Induction of callus and shoot formation were conducted on

solid MS medium (Murashige and Skoog 1962) supplemented with 2.0 mg/l indole acetic acid,

1.0 mg/ benzyl-aminopurine, and 100 mg/l kanamycin sulfate as a selection agent. For root

induction, kanamycin-resistant regenerated shoots were transferred to hormone-free MS

medium containing 50 mg/1 kanamycin. After root development (2 to 3 weeks), plantlets were

transferred to potting soil.

Pokeweed antiviral protein (PAP), a ribsome-inactivating protein, has been recognized

as a broad spectrum virus inhibitory agent. Mutant PAP efficiently inhibited viral gene

expression at both the translational and transcriptional levels without causing host cell toxicity.

Recently, Li and co-auther (2013) was transferred the non-cytoxic pokeweed antiviral protein

(mutant PAP) gene into petunia cells with A. tumefaciens. Forty-two putative transgenic

regenerated lines were obtained from the selected explants. Successful integration of the mutant

PAP gene into the genome of transgenic petunia was confirmed by PCR and Southern blot

analysis. Expression of the PAP gene was further confirmed by RT-PCR and Western blot

analysis. These results were consistent with the assay of resistance to CMV. Fifty-six plants

immune to CMV infection were recovered from nine transgenic lines. Another 51 plants tolerant

to CMV were obtained from 10 transgenic regenerated lines.

2.4.5 Concerns about field release of transgenic plants

Potential risks in the use of VRTPs are essentially similar to those posed by conventional

biotechnology and plant breeding (Varma 1997). However, for achieving acceptance of VRTPs

various concerns must be judiciously addressed on strong scientific basis. Environmental risks

related to the use of VRTPs are not greater than those caused by normal infection of plants by

viruses (Table 14).


50

Table 14 Potential risks in the use of VRTPs for integrated virus disease management.

Potential risk Environmental impact

1-Transfer of transgenes to related species Favorable

2-Development of resistance breaking strains (s) of the virus

Not greater than the use of conventional resistant hosts

3-Transgene of viral origin amy result in emergence of new recombinant viruse which may be more virulent and have enhanced host range

Not greater than the normal evolution of viruses

4-Trans encapsidation may occur Not greater than the normal circumstances

5-Adverse effect of marker genes like the antibiotic resistance genes

Marker genes could have adverse effect, but their use is avoidable

6-Toxicity of transgene produces Transgenes of virus origin are safe as their products are a part of normal diet. Similarly 'R' genes are safe. Other transgene would need appropriate biosafety tests

7-Insertion of transgene into structural gene It would cause phenotypic changes and such VRTP would not be used

8-Loss of biodiversity due to replacement of the traditional varieties

No more than caused by the improved crop varieties in use.

Source: Verma et al. 2002.

Main the possibility about field release of VRTPs is about the possibility of generation

of new viruses/strains as a result of recombination and/or trans-encapsidation. Recombinants

between TMV vector and TMV in TMV transformed N. benthamiana plants have been

observed. PPV with mutated CP gene is able to cause systemic infection in N. benthamiana,

transformed with wild type PPV CP but not in non-transgenic plants, suggesting

complementation of CP gene in transgenic plants. Such events do occur in nature during co-

infection of two viruses of same or different taxon, which occasionally lead to virulent forms as

has been found for geminiviruses. The recombinants must not only be viable but also have some

selective advantage in the transgenic plants. A recombinant arising in transgenic plants will be

inhibited or eliminated by the resistance mechanisms, like PTGS, of the transgenic plant.


51

Although the risks posed by deployment of transgenic technology seem to be small, the

potential risks must be minimized by suitably tailoring the transgene itself. This could be

achieved by (i) use of defective forms of viral genes, (ii) use of untranslatable RNA sequences;

(iii) use of genes from mild endemic isolates, (iv) avoidance of replicase recognition sequences,

and (v) combining/pyramiding transgene with other types of resistance such as plant-expressed

antibodies, antiviral proteins or dsRNA specific nucleases.

Many a times, transfer of transgene to related species is considered a serious risk in the

field use of transgenic plants as some weeds may become weedier and the transgene may move

to non-targeted crop species. Firstly, such flow of genes under natural ecosystem is remote and

secondly, if it does occur it would be environmentally favorable as build up of virus inoculum in

weed plants, which are a major source of virus infection, would be reduced leading to reduction

in epidemics.

As far as the non-target crop species is concerned, this too would be of advantage as

virus infection is not desirable in any cropping system. III effect of transgene on human, animal

and plant health is another area of concern. This should be appropiately addressed when

transgenes are used from non-edible plants and other sources. Overall, the bio-safety concerns in

the use of VRTPs are insignificant. The potential benefits of VRTPs also include reduction in

the use of pesticides for vector control, improved crop quality, possibility of developing

varieties with multiple virus resistance, and decreased seed certification costs. In addition,

VRTPs are important genetic source for plant virus resistance.

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