REVIEW OF LITERATURE - Information and Library …shodhganga.inflibnet.ac.in/bitstream/10603/7047/7/07... · · 2015-12-04Review of Literature 4 ... In fruit crops, viruses are

Review of Literature

4

REVIEW OF LITERATURE

The horticulture industry has experienced tremendous growth in the past 50 years

and has developed into one of the major economic branches of modern agriculture.

Pome [apple (Malus × domestica Borkh.), pear (Pyrus spp.), quince (Cydonia

oblonga)] and stone [plum (Prunus domestica), peach/nectarine (Prunus persica),

cherry (Prunus avium), apricot (Prunus armeniaca), almond (Prunus dulcis)] fruits,

grown mainly in the temperate and sub temperate regions, are important temperate

fruits of the world. Out of all these temperate fruits, apple is the most vital in terms

of production and extent in India and the world (Table 2.1).

Table 2.1: Agriculture statistics of apple in HP, J&K, India and world (2008-2009)

Region Area under

cultivation (ha)

Total Production

(Tonnes)

Productivity

(MT/ha)

Himachal Pradesh *

Apple 97200 5,10,200 5.2

Jammu & Kashmir*

Apple 133700 13,32,800 10.0

India**

Apple 2,74,000 19,85,000 7.24

World**

Apple 47,95,970 6,98,19,324 14.56

*NHB(2009)

**FAO (2008)

India ranks eighth in the world with 20 lakh MT of fresh apple production (FAO,

2008). About 99 percent of India’s apple area falls under the North Western hill

region covering six districts of Himachal Pradesh (HP) (Shimla, Kullu, Sirmour,

Mandi, Chamba, Kinnaur), six districts of Jammu and Kashmir (J&K) (Srinagar,

Budgam, Pulwama, Anantanag, Baramullah, Kupwara) and eight districts of

Uttarakhand (Almora, Nainital, Pithauragarh, Tehri, Pauri, Chamoli, Uttarkashi,

Dehradun). In the North-eastern hills, good quality apple is grown in a small area in

Tawang belt of Kameng district in Arunachal Pradesh and some areas in Nagaland

(Fig 2.1). Due to introduction and adaptation of low chilling cultivars of temperate

crops like pear, peach and plum are now also being grown commercially in certain

areas of the north Indian plains. The apple production data (2008-2009) available for

different states show that J&K and HP account for 90 percent of total apple


5

plantations in India (Table 2.2). Commercial apple cultivation plays a pivotal role in

the economy of growers in hill states of India as sometimes apple is the sole crop.

Any losses to apple will adversely affect the economy of hill farmers.

Table 2.2: (a) Area, production and productivity of apple in different states of India.

Source: National Horticulture Board, Ministry of Agriculture (NHB) (2009),

Government of India. (b) Area, Production and Productivity of apple and other

temperate fruits in India.

State Apple (2008-2009)

Area (000’ha) Production

(000’MT)

Productivity

(MT/ha)

Himachal Pradesh 97.2 510.2 (25.7%) 5.2

Jammu and Kashmir

133.7 1332.8 (67.1%) 10.0

Uttarakhand

32.7

132.3 (6.7%)

4.1

Arunachal Pradesh 10.8

9.8 9 (0.5%)

0.9

Nagaland 0.07 0.1 (0.003%) 1.4

Total 274.47 1985.1 7.2

(a)

*(Anonymous, 2010c)

***(NHB, 2009)

(b)

State

2008-2009

Area (ha) Production

(MT)

Productivity

(MT/ha)

Himachal Pradesh*

Apple 94438

510160

5.4

Other temperate fruits 26546

39930

1.5

Jammu and Kashmir**

Apple 133700 1332800 9.96


6

Fig 2.1: Map of India showing the apple growing areas highlighted in red.


7

In India, apple as a cash crop was introduced by the Britishers in the Kullu valley of

the Himalayan state of HP as far back as 1865, while the colored ‘Delicious’

cultivars of apple were introduced to Shimla hills of the same state in 1917. Among

the indigenous apple cultivars ‘Ambri’ is considered to be native to Kashmir and was

being grown long before Western introductions. Pears and other deciduous fruits

were domesticated successfully in the early part of the 20th century, although some

of them were reported to occur under semi-wild conditions in India much earlier

(Gosh, 2001).

2.1 LESSER PATHOGENS INFECTING APPLE

Like other crops, apple production is hampered due to various abiotic and biotic

stresses resulting poor health of trees and low productivity. It is evident from the

productivity of apple in India which averages to 6-7 MT/ha in comparison to

productivity of 25- 30 MT/ha in leading apple producing countries as small as Italy.

The apple trees are susceptible to a number of fungal and bacterial diseases and wide

range insect pests. Some of the more common diseases/pests of apple in India are

mildew, aphids, apple scab and lately Marssonina blotch (Sharma et al., 2003).

Amongst the various factors responsible for low productivity, infection due to virus

(es) and virus like pathogens have also been found to be a limiting factor in growing

healthy apple orchards. In fruit crops, viruses are important but lesser studied

pathogens particularly in India

Apple is propagated vegetatively by grafting of desired cultivar (scion wood)

on a suitable rootstock which could be an apple seedling or clonal rootstock. Apple

viruses mostly spread through the use of infected budwood in propagation and once a

plant is infected, it cannot be cured by any chemical treatment. The rapid emergence

and marked geographic spread of viruses is due to the enhancement of the

international trade of propagative materials and their final products without proper

quarantine practices.

A number of viruses have been reported from apple growing areas: Apple chlorotic

leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Apple stem pitting (ASPV),

Apple stem grooving (ASGV) (Desvignes et al., 1990; Campbell, 1963; Posnette et

al., 1963; Zahn, 1996), Carnation ring spot virus (CRSV), Cherry rasp leaf virus

(CRLV), Sowbane mosaic virus (SoMV), Tomato ring spot virus (ToRSV), Tobacco

ring spot virus (TRSV), Tobacco necrosis virus (TNV), Tomato bushy stunt virus


8

(TBSV) (Németh, 1986a), Prunus necrotic leaf spot virus (PNRSV) (Sánchez-

Navarro and Pallás, 1997; Chandel et al., 2008b) and Little cherry virus 2 (LChV-2)

(Eppler et al., 2001). Apart from ApMV, ACLSV, ASPV and ASGV rest are minor

viruses and do not cause major economic loss to the yield though apple plant serves

as natural alternative host to them. All the major viruses (ApMV, ACLSV, ASPV

and ASGV) are also the quarantine viruses in most of the countries viz. Australia,

New Zealand and according to EPPO certification schemes. These viruses naturally

infect and cause disease not only apple but in all the pome and stone fruits.

Apple has also been reported to be infected by Apple scar skin viroid

(ASSVd) and Apple fruit crinkle viroid (Koganezawa et al., 1982; Di Serio et al.,

2002; Koganezawa et al., 2003). Viroids as pathogenic agents are limited in range of

crop species they affect compared to viruses (Matthews, 1992). ASSVd directly

affects the apple fruit and causes scarring, dappling and/or blemishes thus affecting

the marketability of the produce. There are many suspected viral and viroid-like

diseases of apple like false sting, green crinkle, dead spur, rough skin, star crack are

suspected to be caused by agents that are graft transmissible however not yet

characterized.

Most of the major apple virus (es), viroids and virus like agents spread due to

infected planting material, i.e. they are graft transmissible. In an experiment by

Zawadzka et al. (1979) ACLSV, ASPV and the apple rubbery wood pathogen were

transmitted to the indicator plants 3-4 days after inoculation with infected bud thus,

indicating that completion of the graft union (8 days) was not to be necessary for

transmission and the failure of graft union did not prevent infection of the stock.

ACLSV, ASPV and ASGV are latent i.e. “symptomless” viruses (Németh, 1986a).

On most commercial cultivars, these viruses remain latent however, may cause

symptoms in certain cultivars.

2.2 LOSSES

The accurate global figures for crop losses due to viruses are unavailable, but it is

generally accepted that losses due to viruses are second only to fungi (Laimer, 2003).

Damage is more profound in perennial crops in comparison to annuals. Viral diseases

cause economic losses through lower yields, reduced quality of plant products and

loss in tree vigour. Most of the viruses can remain latent, spreading through orchards

and inflicting damages. Latent infestations can produce small to moderate losses in


9

fruit production (Agrios, 1997; Cembali et al., 2003). Virus symptoms can be

generally be identified on the leaves as chlorotic alterations, deformations, enations,

necrosis; on fruits as change in shape, color, size, chemical composition and on wood

as pits, difference in diameter between scion and rootstock, graft union necrosis. The

literature reports different types of damages in fruit products, which includes

unmarketable fruits (Reeves and Cheney, 1962), substantial reduction in yields and

extensive tree death (Stouffer and Smith, 1971). Overall viral infections have greater

effect on crop yields and fruit quality (deformation and loss of flavours) than on

vegetative growth. With the most virulent strains the yield losses can reach 98%

(Németh, 1986a). Losses to the quality and quantity of apple produce due to viral

infection have been widely reported from apple growing regions of the world.

Characterization of major apple viruses has just begun in the Indian scenario.

The review presents in brief the work done on ACLSV the virus in focus for the

present study and few other major/ minor virus (es), viroid(s) and graft transmissible

diseases of apple in India and world.

2.3 APPLE CHLOROTIC LEAF SPOT TRICHOVIRUS: VIRUS IN

FOCUS

ACLSV was first reported in Malus spp. from the U.S.A. by Mink and Shay in 1959

(Burnt et al., 1996a). ACLSV synonyms on various hosts are pear ring pattern

mosaic virus (Cropley, 1969), apple latent virus type 1, plum pseudopox virus and

quince stunt virus.

2.3.1 Geographical Distribution

Geographically ACLSV is reported to be worldwide in origin. It occurs probably in

all places where rosaceous fruit trees are cultivated. ACLSV was reported and

described in detail by Luckwill and Campbell (1963) from USA; Cropley (1968a, b);

Lister (1970) from Holland; Németh (1986a) from Hungary; Mink (1989a) from

Japan; Brunt et al. (1996a) from UK; Semenas and Koukharchik (2000) from

Belarus. The occurrence of chlorotic leaf spot disease on Golden Delicious cultivar

of apple was recorded at Regional Fruit Research Station, Mashobra, HP on the basis

of graft transmission of symptoms but nothing was mentioned about the association

of any particular virus (Nagaich and Vashisth, 1965).


10

2.3.2 Host Range and Symptomatology

The importance of ACLSV is due to its worldwide occurrence and its large host

range on pome (apple, pear, quince) and stone (peach, plum, apricot, almond, cherry)

fruit crops which are of great economic value. ACLSV infection has also been

reported on mountain ash (Sorbus aucuparia) by Polák and Zieglerova (1996),

ornamental dwarf almond (Prunus glandulosa Thunb.) by Spiegel et al. (2005),

hedgerow hawthorns (Crataegus spp.), hedgerow blackthorn (P. spinosa) by Sweet

(1980) and on raspberry by Cadman (1970). Among herbaceous hosts Chenopodium

quinoa and Phaseolus vulgaris are local lesion hosts of ACLSV while, C.

amranticolor is the systemic host.

Most commercial cultivars of apple, apricot, cherry, peach and plum do not

exhibit symptoms of ACLSV infection. However, variable symptoms depending on

the virus strain and the host species or cultivar infected are reported. In sensitive

Malus cultivars, symptoms of chlorotic leaf spots and/or ring and line patterns on

foliage, asymmetric leaf distortion, premature leaf drop, stunting, terminal dieback,

inner bark necrosis and xylem pitting, and local bark necrosis surrounding the

inoculum buds have been recorded (Mink, 1989a). Malformation and reduction in

leaf size and chlorotic rings or line patterns were accentuated by ACLSV infection

(Németh, 1986a). The virus caused top working disease of apple trees grown on

Malus prunifolia var. ringo root stocks in Japan (Yanase, 1974; Yanase et al., 1979).

Some ACLSV strains caused russetting and lethal decline of apple on certain

rootstock varieties (Desvignes and Boye, 1989) while, pear ring pattern mosaic on

pear has also been observed (Lemoine, 1977).

In plum ACLSV infection caused bark splitting and mild pox symptoms that

sometimes could be mistaken for plum pox. Symptoms like sunken spots, bands or

rings on the skin of fruit and leaf symptoms have also been observed (Németh,

1986a). Fruit symptoms of pseudopox are difficult to distinguish from those of plum

pox, but leaf symptoms are more distinct. Symptoms on bark include brownish-red

areas on the bark followed by severe cracking, necrosis and splitting. Necrosis of

cambium leading to branch die back has also been recorded. The tree development is

slowed and a vigorous growth of suckers around the tree base has been observed

(Németh, 1986a; Dunez et al., 1972).

In cherry, disease known as 'apple ringspot' is believed to be caused by dual

infections with ACLSV and a severe strain of ASPV. Most varieties of sweet cherry


11

and sour cherry are latently infected by ACLSV, although in some cultivars the

appearance of necrotic, sunken spots on the fruits has been associated with dual

infection by ACLSV and PNRSV (Németh, 1986a). On peach ACLSV often causes

graft-incompatibility, leading to necrosis and early decline. It is also known to cause

dark-green, sunken spots or wavy lines on leaves along with severe fruit and leaf

deformation known as “butteratura” in peach (Németh, 1986a; Šutić et al., 1999b). In

apricot, the virus caused russetting and fruit disease, “viruela” with symptoms

resembling apricot ring pox (Liberti et al., 2005).

2.3.3 Transmission and Vector Relationships

Spread of the virus in the field has been detected however, the natural mode of

transfer is unknown (Brunt et al., 1996). McCrum (1965) concluded that virus could

spread from infected trees of indicator (R 12740-7A) to adjacent healthy indicator

plants through direct mechanical contacts. Shoots which were in direct contact of the

infected plants expressed initial symptoms. Smith (1972) reported transmission of

ACLSV from peach to peach through the contaminated budding knife. No vector has

been identified for ACLSV transmission (Gilmer and Whitney, 1974). Unconfirmed

reports of role of Eudorylamoid nematode in transmission requires further

confirmation (Burnt et al., 1996). García-Ibarra et al. (2010) reported that ACLSV

infection affected the germination of the apricot seeds however, it was confirmed that

ACLSV was not seed transmitted as reported earlier by Šutić et al (1999b).The virus

is thus transmitted by grafting of infected planting material, mechanical inoculations

and unclean horticultural practices.

2.3.4 Losses Due to ACLSV

Of all the viruses infecting apples, ACLSV is the most important virus which causes

huge losses to apple crop (Nemchinov et al., 1995). In nurseries ACLSV induced

severe graft incompatibilities in mostly some Prunus combinations, causing major

problems (Ulubas and Ertunc, 2005). The sensitivity of some apricot seedling

rootstocks to the ACLSV also cause graft-incompatibility (Németh, 1986a; Hansen,

1995a; Liberti et al., 2005, Desvignes and Boye, 1989). Plum pseudopox and apricot

ring pox caused by ACLSV infection also decreases the market value of fruit from

infected trees.


12

In China, Liu et al. (2002) revealed that in top worked apple orchards, 41.7-

88.55% of trees were infected with top work disease resulting in a tree mortality of

15.2-70.0%. Infected trees grew normally in the first 1-2 years, but fibrous and main

roots started to die gradually, shoot growth was limited and fruits were small. The

existence of three latent viruses ASPV, ACLSV and ASGV in the infected trees was

confirmed. Xiang and Zhou (2000) reported 53.06% mortality of topworked apple

trees infected with decline disease caused by ACLSV /ASGV /ASPV. The leaves of

infected trees were small, light yellow coloured and abscission occurred early. These

viruses infect many commercial apple cultivars with an infection rate of up to 80-

100% and cause yield loss of up to 40% in P. R. China (Wu et al., 1998). Cembali et

al. (2003) also reported losses of the order of 30% in fruit yield of Golden Delicious

cultivar of apple due to mix infection of ASGV, ASPV and ACLSV.

Virus infection makes the plant more susceptible to other pathogen attacks

and also reduces the physiological activities, predisposes the tree to nutrient

deficiencies, reduces vigour and in general shortens the life of the plant. Cummins et

al. (1978) recorded the responses of scab-resistant derivatives of Malus species to

infection with ACLSV and other common viruses. One of seven advanced selections

derived from scab-resistant M. floribunda exhibited depressed growth in an orchard

on ACLSV-infected M9 rootstocks compared to growth on virus-free M9. Warner

and Heeney (1985) compared major nutrient levels of ACLSV infected and healthy

McIntosh apple leaf between the 8th and 12th years. Leaf N, P, K and Ca were

reported to be low in inoculated trees.

Another study by Alleyne (1989) regarding water uptake rate measured at 3

levels of suction (40, 53 and 67 kPa) and at 3 temperatures (0, 25 and 35 degrees C

for virus free (ASPV, ASGV, ACSLV) and virus infected rootstocks was conducted.

There was evidence that water uptake rates in MM.106 and MM.111 were higher in

virus-tested than in common clones. Virus-tested rootstocks showed significant

increases in water uptake only between 25 and 35 degrees, with no significant

change below 25 degrees. Arai et al. (1990) observed that apple root stock infected

with ACLSV were more susceptible of to white root rot and violet root rot than the

virus free counterparts.

2.3.5 Virion Properties

2.3.5.1 Particle Morphology and Physiochemical Properties


13

ACLSV particles are very flexuous, filamentous and not enveloped with a clear

modal length of 680-700 nm and 12 nm width with obvious cross banding

(Yoshikawa et al., 1997). For very pure preparations of ACLSV virions sediment as

single or as two very close bands with an S20w of about 100S and electrophoretic

mobility of 5.1 x 10-5

cm2/sec/V in 0.05M Tris + 0.005 M MgCl2 has been reported

(Lister, 1970; Bar-Joseph et al., 1979). In the absence of certain divalent cations or

polyamines, particles degrade, forming fragments sedimenting at about 17S (Lister

and Hadidi, 1971). The UV absorption

spectra of the purified virus preparations had A260/A280 ratio of 1.85-1.89 (Lister and

Hadidi, 1971; Yoshikawa and Takahashi, 1988).

2.3.5.2 Properties in Sap

Lister et al. (1964) concluded that ACLSV had moderate thermal inactivation point

(52-55oC) in Chenopodium quinoa sap with a dilution end point of 10

-4. The

longevity in vitro (LIV) of the virus was reported as 1 day at 20oC or 10 days at 4

oC

(Saksena and Mink, 1969).

2.3.5.3 Biochemical Properties

2.3.5.3.1 Nucleic Acid

ACLSV is unipartite, single-stranded, plus-sense RNA that is polyadenylated with a

total genome size of 7,555 nucleotides excluding poly-A-tail (Lister and Bar-Joseph,

1981; Yoshikawa and Takahashi, 1988; German-Retana et al., 1997). Nucleic acid

constitute 5% of the virion by weight (Lister and Bar-Joseph, 1981).

2.3.5.3.2 Protein

The viral genome encodes structural proteins and non-structural proteins. Virions are

composed of one structural protein a single polypeptide (21.5-28 K). Non-structural

proteins of ACLSV are (1) a protein of about 180-220K containing RNA-dependent

RNA polymerase, helicase and methyltransferase signature sequences typical of

replication-associated proteins of the “alpha-like” supergroup of ssRNA viruses; (2)

a polypeptide of 50K with weak homologies to some plant virus movement proteins

(MP) (Lister and Bar-Joseph, 1981; Bar-Joseph et al., 1979; Yoshikawa and

Takahashi, 1988; German et al., 1990; Candresse et al., 1996). Lipids and

carbohydrate moieties are not reported in ACLSV.


14

2.3.6 Genome Organization

ACLSV is the type species of the genus Trichovirus, family Betaflexiviridae

(Carstens, 2010). In ACLSV the genome is constructed of three slightly overlapping

open reading frames coding for replication-related proteins (ORF 1), a movement

protein (ORF 2), and

the coat protein (ORF 3), respectively (Fig. 2.2). ORFs 2 and 3 are probably

expressed through 3’- coterminal subgenomic RNAs (Fig. 2.3) (German et al., 1990;

Martelli et al., 1994; Yaegashi et al., 2007a). ORF-1 encodes a 216.5 K protein

which contains the conserved signature sequences and has significant homology with

proteins suspected to be involved in viral replication ORF-2 encodes a 50K

movement protein which contains nucleic acid binding domains (Isogai and

Yoshikawa, 2005; Sato et al., 1995). The ORF-2 encoded protein is responsible for

virus cell to cell spread. The 28 K ORF contains, in frame, a smaller 21.5 K ORF

encoding the coat protein (CP) of ACLSV present as multiple copies (German et al.,

1990; Martelli et al., 1994).

The ACLSV-infected tissues contain six dsRNA species of approximately

7.5, 6.4, 5.4, 2.2, 1.1, and 1.0 kbp. The 7.5 kbp species represents the double-stranded

form of the full-length genome, whereas the 2.2 and the 1.1 kbp species are the

double-stranded forms of sgRNAs coding for the putative MP and the CP,

respectively. The most abundant dsRNA species, the function of which are unknown,

are 5’ co-terminal with genomic RNA, and have a size of 6.4 and 5.4 kbp,

respectively (German et al., 1992). Replication is presumed to be cytoplasmic and to

involve the product of ORF1. Ohki et al. (1989) indicated that the viral particles

occur as aggregates in the cytoplasm of vascular parenchyma, mesophyll cells and

rarely in nucleus (Yoshikawa et al., 1997). The complete nucleotide sequences of

ACLSV isolates from apple (Sato et al., 1993a), cherry (German et al., 1997), peach

(Marini et al., 2008) and plum (German et al., 1990; Jelkmann, 1996) have been

determined.

2.3.6.1 Purification

Lister (1970a) and Saksena and Mink (1969) purified the virus from Chenopodium

quinoa by using 250 ml of 0.01M tris-HCl buffer, pH 7.2-7.6, containing 0.01M

MgSO4 or MgCl2, 3-4g of bentonite solution per 100ml of 0.01M phosphate buffer.


15

When centrifuged in sucrose-gradients the virus gave a single light scattering band

(Lister and Hadidi, 1971;

Fig 2.2: Genome organization of ACLSV. MET-methyltransferase; P-PRO papain-

like protease; POL- RNA polymerase and together they form the viral replicase; MP-

movement protein; CP-coat protein.

Fig 2.3: Gene expression in ACLSV. The viral RNA is translated as a

monocistronic mRNA to produce the RdRp (encoded by the 5´-proximal ORF). A

negative-sense complementary ssRNA is synthesized using the genomic RNA as a

template. New genomic RNA is synthesized using the negative-sense RNA as a

template. Internal subgenomic(sg) promoters are used to transcribe the sgRNAs.

Translation of these sgRNAs yields the capsid and movement proteins (German et

al., 1990; Martelli et al., 1994; Yaegashi et al., 2007a)

(http://expasy.org/viralzone/all_by_species/273.html).


16

Lister and De Sequeira, 1969). Lister and Hadidi (1971) showed that by including

magnesium in the extraction buffer degradation of ACLSV was reduced. Dunez et al.

(1973) used an extraction buffer with a high pH (9.5) containing 0.2% 3,3-

diamrnodipropylamine to prevent ACLSV aggregation and fragmentation. ACLSV

was earlier grouped with the Closteroviruses (Lister and Bar-Joseph, 1981) because

of their closterovirus-like particles which often became entangled, clumped together

and fragmented easily (Francki et al., 1985). This had possibly contributed to the

variability in ACLSV particle length reported in the literature (Lister, 1970; Dunez et

al., 1973; Lister and Bar-Joseph, 1981; Thomas, 1983). Thomas (1983) compared

length measurements of ACLSV particles from leaf sap with purified particles and

found that they were 825 nm and 795 nm, respectively. Diethyl ether and carbon

tetrachloride were used for clarification in order to obtain a high yield (l mg/100g

tissue) of purified virus particles. Longer virus particles produced due to end-to-end

aggregation in few cases was also recorded.

Legrand and Verhoyen (1986) used 0.25% formaldehyde in the clarification

step in attempting to reduce viral degradation. While these procedures apparently

decreased fragmentation or increased yield, they reportedly gave inconsistent results

(Dunez et al., 1973; Thompson 1990).

James and Monette (1992) described a reproducible procedure for

purification of ACLSV from C. quinoa using two cycles of sucrose density-gradient

centrifugation in a magnesium-containing buffer. A relatively high yield of virus

particles was obtained with little degradation observed. The average yield was 2.3

mg 100g/l fresh leaf tissue. The modal lengths of purified virus particles and

particles from leaf sap were recorded as 692 nm and 704 nm, respectively.

2.3.7 Detection of ACLSV

2.3.7.1 Biological Detection

Mink and Shay (1959) at Purdue University, Lafayettee, Indiana, reported that

Russian var. R 12740-7A and its progeny proved to be valuable indicators of apple

mosaic and stem pitting viruses and also for a new virus named as chlorotic leaf spot

virus (CLSV) while indexing virus infection in an apple orchard. They found that

most varieties were latent carriers of apple stem pitting and apple chlorotic leaf spot

viruses. Similarly in England, M. platycarpa was reported to induce line pattern

symptoms on leaves following bud inoculation from symptomless commercial


17

cultivars of apple and they named it as Platycarpa line pattern virus (Luckwill and

Campbell, 1959).

Cation and Carison (1960) carried out indexing of apple trees for viral

detection. For this sap inoculation was made from fully expanded young leaves and

inoculated on C. quinoa and C. amaranticolor plants at 3-4 leaf stage. After 4-6 days

of inoculation circular necrotic lesions were observed on C. quinoa and small

chlorotic lesions which become necrotic on C. amaranticolor. After 3-4 weeks of

inoculation of C. amaranticolor leaves on the young plants (6-10 leaf stage) also

resulted in systemic infection with chlorotic spots, rings, lines and distortion

symptoms. Lister et al. (1964) at Department of Botany and Plant Pathology, Purdue

University, USA reported for the first time the back transmission of ACLSV from

systemically infected C. quinoa to woody indicator R 12470-7A by approach

grafting. Hillegonda (1967) from Institute of Phytopathological Research,

Wageningen reported the method of sap inoculation on C. quinoa for rapid indexing

of trees. They concluded that best results can be obtained when homogenated petals

were used as inoculum.

Chairez and Lister (1973a) also obtained symptoms for ACLSV (apple

isolate) on a herbaceous indicator C. quinoa after 3-4 days of inoculation. Virus

related antigens, probably various polymeric forms of viral coat protein, were also

detected in crude extracts from plants infected with ACLSV using highly specific

antisera to two ACLSV strains from apple, peach. The two antisera detected soluble

antigens differently, though with about the same sensitivity (Chairez and Lister,

1973b). Walt and Engelbrecht (1974) while identifying the viruses of pome fruit

trees in South Africa by sap inoculation to C. quinoa and cucumber along with

serological assay reported the wide spread occurrence of ACLSV in Golden

Delicious, Granny Smith and Starking cultivars of apple. Often ASGV and PNRSV

were also observed associated with ACLSV in various apple cultivars. Biological

detection of ACLSV through the use of diagnostically susceptible species like Malus

sylvestris (woody indicator), C. quinoa and C. amaranticolor (herbaceous indicators)

was employed by (Mink, 1989a; Brunt et al., 1996). This method of virus indexing

using indicator plants has also been recommended by international working group on

fruit tree viruses – International Society for Horticultural Science (ISHS)

(Anonymous, 1980).


18

2.3.7.2 Serological Detection

Serological detection can be characterized as quantitative analytical method applied

for measuring biologically important compounds/organisms using antibodies as

specific analytical reagents. These are based on unique recognition reaction between

antibodies and antigens, which elicit their production. Enzyme-labeled antibodies

have been used for some years in the detection of various antigens in tissue sections,

but they are use in quantitative procedures started in nineteen seventies (Engvall and

Perlmann, 1971). Micro plate method of ELISA was introduced for diagnosing

variety of antigens by Voller et al. (1978, 1976a,b). Immunological assay is the

single most important method for disease diagnosis and pathogen detection these

days. It offers great versatility in type of tests and formats used in specific serological

tests (Van Regenmortel, 1982). However, the direct procedure is reported to have

higher specificity for serotype detection and large scale routine testing (Khetarpal

and Maury, 1990; Khetarpal et al., 1990).

2.3.7.2.1 Enzyme-Linked Immuno Sorbent Assay

Till 1977 detection and identification of ACLSV was carried out through biological

assays.

Enzyme-linked immunosorbent assay (ELISA) has been very popular for detection

of viruses in plant material, insect vectors, seeds, and vegetative propagules since it

was introduced to plant virology by Clark and Adams (1977). It was only after this

historic discovery that detection of plant viruses including various reports of ACLSV

detection through ELISA appeared in literature. Clark and Adams (1977) at East

Malling Research Station, Kent, U.K. detected ACLSV in apple leaves by double

antibody sandwich form of ELISA (DAS-ELISA) as described by Voller et al.

(1976b). Pracnos et al. (1981) indexed 534 stone fruit samples for ACLSV by using

biological indicator GF305 Peach seedling and ELISA. In the finding, it was

concluded that both the methods were able to detect the infection but that ELISA was

more reliable as it resulted in detection of virus in more number of samples.

Immunological assay is the most important method for virus diagnosis

these days. It offers great versatility in type of test and format used in specific

serological test (Van Regenmortel, 1982). Serological detection is a quantitative

analytical method applied for measuring biological important compounds or

organism using antibodies as specific analytical reagents. Barbara and Clark (1982)


19

used indirect ELISA to investigate (i) the feasibility of assaying ACLSV in fruit trees

throughout the growing season (ii) distribution of detectable antigen in aerial parts of

the tree (iii) occurrence of different serotypes of virus and the potential for

discriminating among serotypes. They observed that virus was erratically distributed

with leaves towards the base of each branch more often containing virus than those

towards the tip. Bark stripped from 1 or 2 years old wood was the most reliable tissue

for assay, particularly later in the growing season. Discrimination among virus

serotypes was done by testing each isolate with two distinct antisera, one with

specificity for apple isolates and one with broad spectrum specificity.

Fuchs (1983) at Martin Luther University, Halle Germany, compared the

different serological methods for detecting ACLSV in apple. He recommended the

latex text for detection of ACLSV in petals of apple flower however, ELISA had

been reported to be reliable in the detection of this virus in forced leaf buds and

peals. An improved test calendar for serological detection of the virus in apple was

also highlighted in his findings. Rankovic and Vuksanovic (1983) investigated the

detection of ACLSV by ELISA technique in different plant parts of 40 apple

cultivars grafted on different rootstocks and seedlings. ACLSV was detected in buds,

leaves, petals and fruits of all cultivars grown in Yugoslavia as standard grafting

material. However, it was absent only in some indigenous varieties and one variety

of foreign origin grafted on seedling. Authors also obtained good results of ELISA

with the addition of 2 per cent and 1 per cent PVP and 0.1 per cent 2-

mercaptoethanol in the extraction buffer. Adams et al. (1984) detected the ACLSV in

apple trees through F (ab1)2 based ELISA technique. They reported that all isolates of

the virus from apple were of similar serotype F (ab1)2 based ELISA test and leaf

samples from near the base of shoot formed in the current season were seemed most

likely to be containing detectable virus. ELISA is being routinely used in the

indexing, certification and quarantine programmes of different temperate fruits in

developed countries for many years (Németh, 1986a).

Workers have used ELISA for preliminary detection of ACLSV and other

related viruses, for estimating the disease incidences and the mixed viral infections in

various other pome and stone fruits (Cambra et al., 1982; Lla´cer et al., 1986, 1997;

Savino et al., 1995). In Bucharest Minoiu et al. (1990) standardized DAS-ELISA to

detect ACLSV the virus in apple, pear, plum and cherry trees in the growing season

while a modified ELISA was also used to detect ACLSV in buds and shoots of plum


20

trees in winter. Polák et al. (1997) from Czeck Republic reported detection of

ACLSV by the use of ELISA in apple and pear orchards. They also studied the

distribution of ACLSV in Czeck Republic and recommended the ACLSV free graft

material for viral elimination programme. The effect of sampling time and plant part

reliability for ACLSV detection in apricot by ELISA was studied by Varveri and

Bem (1997). They recommended leaf as the best source for detecting ACLSV. It was

also concluded that ACLSV could be detected throughout the growing season but

virus titre was highest during March-April and October-November. Lessa et al.

(1998) detected ACLSV in 47.4% of the 116 analyzed samples, affecting 5 of 6

orchards in Brazil. Yaqin et al. (1998) compared three ELISA methods viz. protein A

Sandwich (PAS) ELISA, DAS-ELISA and modified DAS ELISA and concluded that

DAS-ELISA detected the virus in shortest time period. A report by Myrta et al. from

Albania in 2004 demonstrated the high infection rate of ACLSV in pome fruits-

100% in apple and 84.2% in pear. ACLSV generally occurs in mixed infection with

other apple viruses viz. ApMV, ASGV, ASPV (Van der Meer, 1976; Klerks et al.,

2001; Kundu and Yoshikawa, 2008). Many workers have used Caglayan et al. (2006)

concluded that among the mixed infections, the most common one was

ACLSV+ASPV (84.21%), followed by ASPV+ASGV (36.84%), ACLSV+ASGV

(26.32%) and ASPV+ApMV (5.26%). The incidence of the ASPV+ ASGV+ACLSV

combination was 26.32%.

In the Indian scenario, ACLSV was detected in the many commercial

cultivars from various orchards of different apple growing belts of HP using

biological and serological (ELISA) indexing techniques (Thakur and Handa, 2000).

2.3.7.2.2 Antibody development

Stable hybridoma cell lines secreting monoclonal ACLSV antibodies were developed

by Poul and Duenz (1989) against P863 ACLSV plum strain. In 1990, Poul and

Dunez further produced 13 monoclonal antibodies (Mab’s) and tested their

specificity by ELISA. Epitope specificity studies showed that these Mab’s defined in

ACLSV particles seven independent antigenic domains, representing at least 8

distinct domains. It appeared that the interaction between a Mab and the virus could,

in some cases, induce conformational changes in the viral particles which enhanced

the binding of others.


21

Polyclonal ACLSV antiserum has been produced from purified virus obtained after

multiplication in herbaceous host (Fuchs and Merker, 1985; Hong and Wang,

1999).The purified fusion protein has also been expressed and used as an antigen for

obtaining polyclonal antibodies for an ACLSV pear isolate in China (Cai et al.,

2005).

2.3.7.2.3 Electron Microscopy

Methods based on electron microscopy viz. immuno-electron microscopy (Kerlan et

al., 1981) and with colloidal gold staining (Himmler et al., 1988) were used for many

temperate fruit viruses. Gualaccini et al. (1981) reported the presence of ACLSV in

fruit trees from Tuscany, Italy on the basis of symptoms on herbaceous hosts, particle

morphology and immunosorbent electron microscopy (ISEM) in apricot, pear and

plum myrobalan (Prunus cerasifera) in the nursery plants. Kerlan et al. (1981) from

Bordeaux, France reported the use of ISEM for detection of ACLSV in leaf extracts

of infected peach, plum and apricot in addition to herbaceous host C. quinoa. These

workers found that ISEM was as sensitive as ELISA in the detection of ACLSV and

could be used as a reliable alternate. Savino et al. from Italy in 1991 reported the

identification of different viruses including ACLSV in apricot by ELISA and

immuno electron microscopic (IEM) procedures. Crystallized aggregates of ACLSV

particles as parallelograms and as a circle (0.5- 1.5µm diameter) in the cytoplasm of

chlorotic areas C. quinoa were reported by Ohki et al. (1989).

2.3.7.3 Molecular Detection

With the advent of nucleic acid based molecular detection techniques there has been

a shift

towards the use of polymerase chain reaction (PCR) based techniques for the

characterization and detection of viruses and other pathogens of similar nature. PCR

is a method of in vitro amplification of template DNA sequence with very high

specificity and fidelity using dNTPs, specific primer and Taq DNA polymerase in a

simple automated reaction (Saiki et al., 1985; Mullis, 1990). This enzymatic

amplification of the DNA sequence (by PCR) has increased the sensitivity level of

the test to 10 fg of purified viral RNA (Wetzel et al., 1991).


22

2.3.7.3.1 RNA Isolation

A critical step for routine use of PCR technology is template isolation. The standard

sample extraction procedure for RT-PCR detection of ACLSV is based on nucleic

acid isolation (total RNA) from different hosts. Tissues from woody plants (pome

and stone fruit crop), especially when field-grown could contain higher amounts of

phenolic compounds and polysaccharide thus causing difficulty in isolating total

RNA from these plants in good quantity and quality. These phenolic compounds

from plant tissues inhibit reverse transcription (RT)-PCR (Nassuth et al., 2000; Singh

et al., 2002). MacKenzie et al. (1997) employed commercially available spin-

column matrices and mitigated the inhibitory effects of plant polysaccharides and

polyphenolic compounds commonly observed on subsequent PCR amplification

when conventional extraction methods were applied to woody plant species. The

method has been successfully used in the development of highly sensitive RT-PCR

technique for the detection of a number of viruses in their woody hosts. Further, it

was observed that detection of viral RNA in samples of total plant RNA prepared

using this method was as sensitive as previously described for the immune capture

RT-PCR (IC-RT-PCR) technique.

Singh and group (2002) demonstrated that the adding 0.65 to 0.70% sodium

sulfite in extraction buffer minimized the pigmentation of nucleic acid extracts and

improved RT-PCR detection of viruses from potato tubers and stone fruits. It was

also observed that the resultant nucleic acid extracts were suitable for both duplex

and multiplex RT-PCR. Potentially improved sample processing procedures for plant

virus RNA extraction and subsequent detection by PCR have been reported (Choi

and Ryu, 2003; Foissac et al. (2001). Ruan (2004) demonstrated a method based on

Silica capture without using organic solvent such as phenol and chloroform. The

method was very efficient, less time consuming and decades of samples could be

extracted in 2 hrs.

2.3.7.3.2 Nucleic Acid Hybridization

Hybridization of the total RNA/viral genomic RNA from the infected plant species

with the radiolabeled or non-radiolabeled virus specific DNA/RNA probe is a very

powerful tool for the detection and identification of virus from the infected plants,

because of its very high efficiency and sensitivity. It can also be used for serotype


23

differentiation. Combining PCR with molecular hybridization further increases the

sensitivity of detection of plant pathogens (Vunsh et al., 1990; Borja and Ponz,

1992). Nucleic acid hybridization is a modern method of plant virus and viroid

detection based on identification of specific molecule components of the causal

agents in tested samples. The genetic material of the pathogen can be detected by

nucleic acid hybridization. This technique was initially used in phytopathology for

viroid detection (Owens and Diener, 1981). Considerable progress has been made in

the nucleic acid hybridization, which seems to be a good alternative to ELISA

technique, when virus-specific antiserum is not available or pathogen specific protein

is not produced is host plant.

Tissue print hybridization is also used extensively for detection of viruses.

The most common procedure is the dot blot or slot-blot hybridization. Printing plant

tissue directly to membrane was first reported by Cassab and Varner (1987) and

subsequently the method has been modified to suit different plant species. This

method has the added advantage of being able to localize virus within the plant

(Mansky et al., 1990; Chia et al., 1995). Immuno-tissue printing protocols for the

localization of ACLSV, ASGV and Plum pox virus (PPV) in shoots of Prunus and

Malus spp. in vitro has been established (Knapp et al., 1995a) The ACLSV presence

was checked by immuno tissue printing, DAS and DAC ELISA from the shoots of

prunus and apple maintained in vitro (Knapp et al., 1995a). They found that

accumulation of ACLSV was highest at the base of the stem and decreased toward

the apex of shoots. ACLSV was found in the epidermis, cortex, and vascular bundles

but seldom in the pith tissue of in vitro apple shoots. Wang et al. (1998) reported the

presence of ACLSV and ASGV in the plant extracts through dot-immuno binding

assay (DIBA). Dominguez and group in 1998 used nonisotopic molecular

hybridization techniques for the detection of ACLSV and Ilarviruses in apricot trees

in Spain.

2.3.7.3.3 Reverse Transcription Polymerase Chain Reaction (RT-PCR) / Immuno

capture RT-PCR (IC-RT-PCR)

In 1990 Hadidi and Yang first utilized RT-PCR technique for detection of RNA plant

viruses from infected tissue and predicted the application potential of PCR

technology in the field of plant pathology. PCR was reported to be more sensitive

than direct probing or serological techniques for detecting and characterizing plant


24

pathogens (Hadidi et al., 1995). Print and spot capture polymerase chain reaction

(PCR), heminested PCR and PCR-ELISA are recently developed techniques used for

the detection and characterization of the viruses (Cambra et al., 1998; Candresse et

al., 1998). In case of mixed infections, multiplex PCR and non-isotopic molecular

hybridization has also been used (Menzel et al., 2002, Saade et al., 2000). The

number of plants infected with any viruses was higher when tested using RT-PCR

comparing to ELISA. RT-PCR has been used extensively for the detection of plant

viruses. The technique is very sensitive and is able to detect even picograms of virus.

Candresse et al. (1995) developed a sensitive polyvalent PCR based assay

to detect ACLSV by introducing an additional immuno capture test in the PCR tube.

The results were compared with ELISA and increased rate of ACLSV detection by

immuno capture (IC)-PCR was obtained. In 1995 Nemchinov et al. reported the

detection of ACLSV in apple and peach tissues by using 3 types of PCR viz. RT-

PCR, IC-RT-PCR and multiplex IC-RT-PCR. Nassuth et al. (2000) standardized

simultaneous detection of RNA of ACLSV and mRNA of two plant genes, Malate

dehydrogenase (MDH) and Ribulose Bisphosphate Carboxylase Oxygenase

(RUBISCO) which were used as the internal controls to check false negatives.

Menzel et al. (2002) used multiplex RT-PCR assay for the simultaneous detection of

four apple viruses viz. ASGV, ACLSV, ASPV and ApMV.

After hybridization of the PCR products to specific capture oligonucleotides

anti digoxigenin antibodies were used for detection. The real time 5' nuclease RT-

PCR assay with fluorescent 3’ minor groove binder-DNA probe for detection of

ACLSV from the leaf tissues of apple was standardized by Salmon et al. (2002). This

method combines both the PCR amplifications and DNA hybridization in a single

tube. A sensitive and reliable multiplex RT-PCR-ELISA technique for the detection

of ACLSV, ASGV, ApMV in which the amplified products were labeled with

digoxigenin during the RT-PCR by incorporation of a digoxigenin labeled primer

was developed Menzel et al. (2003). Deng et al. (2004) used immunocapture (IC)

and tube capture (TC) RT-PCR for the detection of ASGV and ACLSV in Pyrus

pyrifolia samples. It was concluded that compared to conventional RT-PCR, IC-RT-

PCR and TC-RT-PCR showed greater sensitivity and simplicity. Simultaneous

detection of ACLSV and five other viruses infecting stone and pome fruits by non-

isotopic molecular hybridization using a unique riboprobe or polyprobe (using

tandemly fused six respective viral sequences) was developed (Herranz et al., 2005;


25

Pallas et al., 2005). On similar lines Foissac et al. (2005) developed polyvalent

degenerate oligonucleotides RT-PCR for polyvalent detection and characterization

for Trichoviruses, Capilloviruses and Foveaviruses. Hassan et al. (2006) made

attempts to detect in a single tube four viruses viz. ACLSV, APMV, ASGV, ASPV

using four virus specific primer pairs. The results were later analyzed with ELISA

and bioassays.

From India ACLSV was recently characterized from apple, wild Himalayan

cherry, almond, quince, apricot and peach (Rana et al., 2007b; 2007a; 2008a; 2008b;

2008c; 2009 respectively). ACLSV infection on plum and pear at molecular level has

also been confirmed (Ferretti et al., 2010; Rana et al., 2010).

2.3.8 Variability Studies

Coat protein of plant viruses determines virus antigenic property. It is also

responsible for virus-vector relationship and their mode of transmission. Variability

in coat protein can lead to change in antigenic property of a virus which can also

change virus-vector relationship. Severe cases of addition and deletion can leads to

evolution of a new strain of virus. Sequence identity of coat protein gene can also be

used as a criterion for taxonomic classification and for phylogenetic studies.

Sato et al. (1993a) compared the complete nucleotide sequence of genome of

ACLSV apple isolate with other sequences of ACLSV available from Gen Bank and

observed 79.8% sequence identity with the ACLSV plum isolate. The coat protein

gene of ACLSV apple and plum isolate shared sequence identity of 88.6 per cent at

amino acid level. Comparison of the coat protein gene of Grapevine berry inner

necrosis virus (GNIV) with coat protein gene of ACLSV and other Trichoviruses

pointed to substantially higher nucleotide and amino acid homology (Minafra et al.,

1994; 1997). Studies on biological, morphological and serological properties of

GNIV the casual virus of grapevine berry inner necrosis disease occurring in Japan

with those of several known Trichoviruses showed that host range, particle length

and coat protein gene of GINV were quite similar to those of ACLSV (Yoshikawa et

al., 1997)

Candresse et al. (1995) observed the molecular variability of ACLSV by

homology search with sequences from Gen Bank and reported that most coding

differences were observed in the putative viral movement protein while coat protein

showed better conservation. Malinowski et al. (1998) and Cieślińska et al. (1995)


26

reported about difference in coat protein properties of an ACLSV plum isolate SX/2.

The coat protein of this isolate migrated faster in SDS-PAGE and did not react with

some monoclonal and polyclonal antibodies prepared against ACLSV isolates from

apple and cherry. Amplification of full coat protein gene was not obtained with

primers from isolates of apple and cherry. Among the isolates of plum, SX/2 showed

85% nucleotide sequence identity with ACLSV P-863 and ACLSV-P-205 isolates

while sharing 93% and 91% identity at protein level respectively.

Analysis of partial nucleotide sequence of CP gene of 35 ACLSV isolates by

Al-Rwahnih and workers (2004) revealed that some isolates from apricot and peach

(Group B) showed great sequence variability throughout the gene, while rest of the

isolates vary among themselves slightly in N-terminal and while the C-terminal is

mostly conserved. However, later Liberti et al. (2005) confirmed that members in

group B were in fact Apricot pseudo-chlorotic leaf spot virus (APCLSV) isolates,

with partial CP amino acid sequences 88 to 97% identical to the APCLSV isolates.

Serce and Rosner (2006) characterized ACLSV isolates from various hosts and

geographic locations in Turkey at molecular level by RFLP. Based on nucleotide

sequence alignment and the phylogenetic tree, they proposed a classification of

ACLSV isolates in which isolates were divided into three major groups. The first

group contained mainly Far-Eastern isolates, the second group the Hungarian

(eastern-European) ACLSV isolates, and the third group, which contained isolates of

variable characteristics, was again divided into two subgroups, subgroup I containing

mixed European isolates, and subgroup II containing central European isolates.

Three representatives Turkish ACLSV isolates belonged to the third group; of these,

one was from the mixed European cluster (subgroup I) and two from the central

European cluster (subgroup II). A correlation between nucleotide sequence

divergence and geographic origin of the ACLSV isolates was proposed.

A classification based on co variation of the five amino acids at positions 40,

59, 75, 130 and 184 which were highly conserved within each cluster was proposed

by Yaegashi et al. (2007a). They designated the isolates containing the combination

alanine 40, valine 59, phenylalanine 75, serine 130 and methionine 184 as ‘P205

type’ while, the isolates containing serine 40, leucine 59, tyrosine 75, threonine 130

and leucine 184 combinations as ‘B6 type’. Agroinoculation assay indicated that the

substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in

extreme reduction in the accumulation of viral genomic RNA, double-stranded


27

RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting

that the combinations of the two amino acids at positions 40 and 75 are important for

effective replication in host plant cells (Yaegashi et al., 2007a). Yaegashi and

Yoshikawa (2010) further examined the stability of mutant CP with an amino acid

substitution (CPm40; Ala to Ser at position 40, CPm75; Phe to Tyr at position 75;

which is fatal to viral infectivity and replication) by agroinfiltration in Nicotiana. The

results showed showed that there were two conflicting roles of CP related to the

ACLSV replication cycle. (1) stable accumulation of CP is important for effective

viral genomic RNA accumulation, and (2) transient expression of CP inhibits viral

genomic RNA accumulation. It was proposed that ACLSV replication may be

regulated by the level of CP accumulation and/or the timing of CP expression.

2.3.9 Expression studies of ACLSV

German et al. (1992) isolated 6 types of ds RNA’s (7.5 kb, 6.4 kb, 5.4 kb, 2.2 kb, 1.1

kb and 1 kb) of ACLSV and reported that 50 K movement protein and 25 K coat

protein was produced by 2.2 kb, 1.1 kb RNAs respectively. A model for expression

of genome of ACLSV was presented. Sato et al. (1993a) cloned and expressed the

full length cDNA of ACLSV, downstream of Cauliflower mosaic virus (CaMV) 35S

promoter and obtained polypeptides of size 190, 60, 56, 22, 15K. Genomic RNA of

ACLSV was translated in a rabbit reticulocyte lysate system which yielded

polypeptides of size 190, 60, 56, 22, 15 k. The 22 k product was immuno precipitated

and identified as coat protein gene (Candresse et al., 1996). Mechanical and biolistic

inoculation of a full length infectious cDNA clone of ACLSV genome was done on

C. quinoa and apple plants for analyzing viral gene function and assignment of

biological properties to viral genes (Satoh et al., 1999).

2.4 APPLE MOSAIC ILARVIRUS

Apple mosaic virus (ApMV) (family Bromoviridae, genus Ilarvirus) present world-

wide and is an economically important and common pathogen in commercial apple

cultivars (Mink, 1989b). It preferentially infects woody hosts such as blackberry,

raspberry (Rubus sp.), apricot, cherry, almond, (all Prunus sp.), roses (Rosa),

mountain ash (Sorbus aucuparia), horse chestnut (Aesculus hippocastanum), red

horse chestnut (A. x carnea) and hop (Humulus lupulus) (Brunt et al., 1996b). ApMV

has also been reported from hazel nut (Corylus avellana) (Aramburu and Rovira,


28

2000) and strawberry (Tzanetakis and Martin, 2005). Synonyms of ApMV are e.g.

European plum line pattern virus, Mountain ash variegation virus, Birch line pattern

virus, Birch ringspot virus, Dutch plum line pattern virus, Hop A virus,

Horsechestnut yellow mosaic virus, Rose mosaic virus, Hop virus A, Hop virus C,

Mild apple mosaic virus and Severe apple mosaic virus.

ApMV possesses a tripartite, positive-sense, single-stranded RNA genome

(Fig 2.4) encapsulated by a coat protein (CP) of approximately 25-30 kDa. It is a

member of subgroup III of the Ilarvirus genus in the Bromoviridae family (Alrefai et

al., 1994). Particles are isometric and two sizes have been reported, c. 25 and 29 nm,

corresponding to two peaks in density gradient centrifugation (De Sequeira, 1967).

RNA 1 and 2 are monocistronic and encodes for structural proteins whereas RNA 3

is bicistronic and encodes for MP and CP, but CP is not translated from RNA 3. A

subgenomic RNA (RNA 4) is also present in case of ApMV which is collinear with

the 3’ end of RNA 3 and encodes for CP. The complete nucleotide sequence of

ApMV has been characterized from several parts of the world. Alrefai et al. (1994)

deduced the sequence of RNA 4 of ApMV for the first time. In 1995, Shiel et al.

deduced the nucleotide sequence of ApMV RNA 3. Shiel and Berger (2000)

characterized the complete nucleotide sequences of ApMV RNA 1

and 2. Sánchez-Navarro and Pallás (1994) proposed the secondary structure for the

3'-terminal region of RNA 4 shows the presence of three hairpin structures flanked

by the tetranucleotide AUGC that are highly similar to those previously described in

the RNA 4 species from Alfalfa mosaic virus (AMV) and Tobacco streak virus

(TSV). The features (metal-binding domain and highly conserved hairpin structures)

are characteristics of ilarviruses and are probably involved in the peculiar 'genome

activation' phenomenon i.e. requiring the presence of CP to initiate infection (Bol et

al., 1971; Jaspars, 1985). The CPs of several ilarviruses are interchangeable and can

activate each others' genome (Van Vloten-Doting, 1975; Gonsalves and Garnsey,

1975; Gonsalves and Fulton, 1977; Van Vloten-Doting and Jaspars, 1977).

ApMV is similar to PNRSV in particle characteristics and host range, but it is

not known to spread through either pollen or thrips like PNRSV (Alrefai et al.,

1994). ApMV in nature is transmitted through root grafting (Hunter et al., 1958),

most frequently through infected planting material (Šutić et al., 1999c). The virus is

not transmitted by Cuscuta campestris, C. gronovii, C. subinclusa (Fulton, 1952) or

C. reflexa (Nagaich and Vashisth, 1963).


29

Apple mosaic characterized by symptoms of mosaic, mottling as well as

necrotic ringspot is one of the oldest known and most widespread diseases caused by

ApMV commonly on apple and rose. The virus is known to cause line pattern

symptoms in plum and silver birch (Betula pendula) (Gotlieb and Berbee, 1973).

Postman and Cameron (1987) reported latent and symptomatic (chlorotic ring spots

and line patterns) infection of ApMV in filberts crop (Corylus Avellana). Most

commercial apple cultivars are known to be affected by ApMV, but vary in severity

of symptoms. Cultivars 'Golden Delicious' and 'Jonathan' are severely affected (Šutić

et al., 1999c), whereas 'Winesap' and 'Mclntosh' are only mild symptoms. Except in

severe cases, infected trees can still produce a crop and yield reductions may vary

from 0 to 50 per cent. ApMV infection in some cultivars greatly affects bud set

(Chamberlain et al., 1971).

The presence of ApMV is also reported to reduce the growth of apple trees

(Chamberlain et al., 1971), increase the height of the climacteric, decrease the

content of malic acid (Makarski and Agrios, 1973), decrease trunk girth (Thomsen,

1975; Šutić et al., 1999c), decrease bud take by 3-20%, reduce the quality and

quantity of pollen (Lemoine, 1982) in infected apple trees and cause severe stunting

in the survived plants (Rebandel et al., 1979). Posnette and Cropley (1956) also

reported can reduction in plant growth by 50%, trunk diameter by 20% and fruit

yield by 30% in ApMV susceptible cultivars. Severely infected plants show

yellowing of leaves, veins and surrounding leaf lamina. Trees infected with ApMV

developed pale to bright cream spots on spring leaves. These spots may become

necrotic after exposure to summer sun and heat. A study on the economic

implications of a virus prevention program in deciduous tree fruits in the USA

reported that ApMV infection alone in Golden delicious cultivar of apple can cause a

yield loss of upto 46 % (Cembali et al., 2003).

The virus is moderately immunogenic; rabbits receiving twice-weekly

intramuscular injections of about 1mg virus emulsified in Freund’s incomplete

adjuvant developed antibody titres of 1/1280 or more after 4 weeks (Fulton, 1967).

The virus reacted well in agar diffusion tests; in liquid precipitin tests precipitates are

granular. Clark et al. (1976) used ELISA for successful detection of ApMV from the

fruits, flowers, leaves and roots of plum. The sensibility of technique and its

suitability in handling many small samples of tissue were exploited in assessing

differences in virus content with in leaves and between different plant parts. Casper


30

(1983) developed antisera against viruses that could detect three viruses viz. Hop

virus, PNRSV and ApMV. Torrance and Dolby (1984) detected the ApMV from

apple trees and reported that the virus is evenly distributed in all the plant parts. They

also reported that freezing was less reliable source for storage of the virus.

Both serological (like ELISA) and molecular based (like RT-PCR,

hybridization etc.) detection techniques have been widely used for the detection of

ApMV in various stone and pome fruit crops including apple (Barba, 1986; Turk,

1996; Petrzik and Svoboda 1997; Piskornik et al., 2002). ApMV is reported to be

serologically related to PNRSV (Casper, 1973). A selection of stone fruits, apple,

hops and roses were tested for presence of ilar viruses including ApMV by Johnstone

et al. (1998). It was reported that isolates from hop were serologically closely related

to ApMV. Posnette and Cropley (1956) reported that mild strains of the virus protect

against more virulent strains in apple.

A comparative study was made (Imed et al., 1997) of the biological,

physicochemical and serological properties of 9 isolates of ApMV recovered from

almond (5), cherry (2), and one each from peach and apricot trees showing different

disease symptoms in southern Italy. In the serological investigations, monoclonal

antibodies raised to an almond virus isolate (ApMV-A11) were used. However,

differences in biological behaviour of 9 ApMV isolates from different Prunus

species could not be linked to any differential physicochemical or serological

property.

Petrzik and Lenz (2002) characterized complete CP of eight ApMV isolates

from almond, apple, hop, prune, and pear. They observed that two American and two

European isolates had insertions 6 to 15 nucleotides after nucleotide position 141.

The insertion resulted in the American isolate an inframe shift repaired with two-

point insertions 17 and 68 nucleotide downstream. The predicted folding of the

translated protein was not influenced by the insertions or frameshift. It was

speculated that the region after nucleotide position 141 was without reasonable

selection pressure and a hot spot for the accumulation


31

Fig 2.4: Segmented, tripartite linear ssRNA (+) ApMV genome composed of

RNA1, RNA2, RNA3. Each genomic segment has a 3’ tRNA-like structure and a

5’cap. ( http://expasy.org/viralzone/all_by_species/136.html)

Fig 2.5: Linear ssRNA (+) ASGV genome of 6.5-7.5 kb in size. The 3’ terminus is

polyadenylated. ORF2 protein (MP) is translated by subgenomic RNA. Capsid (CP)

protein may be produced by cleavage of ORF1, but expression by a subgenomic

RNA (http://expasy.org/viralzone/all_by_species/267.html)

Fig 2.6: Linear ssRNA(+) ASPV genome of 8.4-9.3 kb in size. The 3’

terminus is polyadenylated and 5’end is capped.

(http://expasy.org/viralzone/all_by_species/269.html)


32

of insertion mutations in ApMV. Nonisotopic molecular hybridization and multiplex

reverse-transcription polymerase chain reaction (RT-PCR) methodologies were

developed that could detect the three most economically damaging ilarviruses

affecting stone fruit trees on a worldwide scale i.e. PNRSV, Prune dwarf virus

(PDV), and ApMV simultaneously (Saade et al., 2000). ApMV detection methods

based on RT-PCR have been reported by Choi and Ryu (2003), Crowle et al. (2003)

and Petrzik (2002). ApMV was detected in pears, a previously non-reported virus

host by Peterzik (2005). All nine newly sequenced ApMV isolates from pears had a

15-nucleotide insertion in the capsid protein gene in identical position of that of

apple isolates compared with isolates from hop and prunes. The insertion was the

most prominent (but not essential) modification of the CP gene, which results in a

phylogenetic separation of ApMV isolates into three clusters. Sequence analysis data

of an additional 15 isolates revealed a sequence correlation with kernelled fruit trees

(apple and pear). Saade et al. (2000), Menzel et al. (2003) and Sánchez-Navarro et

al. (2005) standardized multiplex RT-PCR detection of ApMV with some viruses

from apple and some other host tissues.

The ApMV concentration in an infected apple tree varied through the year

similarly to other apple tree viruses such as ACLSV and ASGV, and was higher in

the first half of the year (Fuchs 1982; Matic et al. 2008). ApMV was reported to be

partially systemically distributed in woody hosts (Fuchs and Grüntzig, 1994). Among

the recent advances Lenz et al. (2008) designed an oligonucleotide microarray for

detection of some fruit viruses (ApMV, ASPV, PNRSV, PPV and PDV) and studied

the theoretical detection limit using Cy3-labelled oligonucleotides. The optimal

conditions for detecting ApMV were assessed by Svoboda and Polák (2010) by

determining relative concentrations of viral coat protein in different tissues (leaves,

flower petals, dormant buds, and phloem) in five selected symptomless ApMV-

infected apple trees of two cultivars at different terms during the vegetation period.

Results showed that highest relative virus concentration and therefore the highest

reliability of virus detection was obtained with young leaves in April before

flowering. It was also observed that relative concentration of ApMV in young leaves

and flower petals reached its highest level in spring in the Czech Republic suggesting

that the virus propagated better in colder weather. A fast and simple alternative

detection method with one tube RT-PCR to minimize the time and labour required


33

for the diagnosis of ApMV in hazelnut from various tissue (flower, leaf, husk) was

standardized by Akbas and Degirmenci (2010).

Natural transmission of ApMV through root grafting was established by

Dhingra (1972). Singh et al. (1979) observed that shoot growth, fruit set, fruit

weight, yield/tree and fruit ascorbic acid content of ApMV infected apple trees were

reduced in comparison to virus free plants. Comparative studies were made of 30

year old ApMV infected and healthy apple trees. They reported that shoot growth,

fruit set, fruit weight, yield/tree and fruit ascorbic acid content were reduced by

ApMV infection. Bhardwaj et al. (1994) detected of ApMV in apple using indirect

ELISA from HP, India. Later in 1994 Bhardwaj et al. standardized alkaline

phosphatase (ALP) and penicillinase (PNC) based indirect ELISA for the detection

of ApMV from HP, India. Recently, Thockchom et al. (2009) have detected ApMV

by ELISA and slot blot hybridization in apple, plum and apricot. The ApMV-CP was

also characterized at molecular level from apples.

2.5 APPLE STEM GROOVING VIRUS

Apple stem grooving virus (ASGV) is another latent and economically important

virus in commercial apple cultivars (Németh, 1986a; Welsh and Van der Meer,

1989). It was reported in Malus sylvestris cv. Virginia Crab, from the U.S.A. by

Lister et al. (1965). ASGV is a flexuous filamentous particle of 600-700 x 12 nm in

size (Hirata et al., 2003). The genome is unipartite RNA and contains a

polyadenylated, positive sense, single-stranded RNA of 6,496 nucleotides

(Yoshikawa and Takahashi, 1988; Yanase et al., 1990; Yoshikawa et al., 1992). The

ASGV genome consists of two overlapping open reading frames (ORFs) (Fig 2.5)

encoding a 241-kDa polyprotein and a 36-kDa protein (Yoshikawa et al., 1992). The

241 kDa polyprotein contains the conserved motifs of a helicase, a RNA polymerase

and the coat protein coding region. Another important member of the Capillovirus

group is Cherry virus A (CVA) (Adams et al., 2004).

ASGV has been reported to be seed-transmissible in apple (Malus

platycarpa) (6%) (Šutić et al. 1999a; van der Meer 1976), lily (Lilium longiflorum)

(2%) and C.quinoa (2.5–60%) (Inouye et al. 1979). However, seed transmission of

ASGV in pear has not been found. Shim et al. (2006) indicated that ASGV could be

transmitted by a fungus Talaromyces flavus to pear (20% infectivity) and P. vulgaris

(35–90% infectivity) plants by direct infiltration into leaves with ASGV infected T.


34

flavus. The pear and P. vulgaris plants inoculated with ASGV-infected T. flavus

developed similar symptoms of black necrotic leaf spot and chlorotic spots,

respectively, as those observed in plants which were mechanically inoculated with

the crude saps from ASGV-infected C. quinoa.

The host range includes apple (Jones and Aldwinckle 1990; Magome et al.

1997; Nickel et al. 2001), pear (Jones and Aldwinckle 1990; Yoshikawa et al. 1996;

Shim et al. 2004; Wu et al., 2010), apricot (Takahashi et al., 1990; James, 1999) and

cherry trees (Kinard et al., 1996). ASGV has been reported to infect citrus (Lovisolo

et al. 2003; Magome et al. 1997), lily (Inouye et al. 1979), and kiwifruit (Clover et

al. 2003). ASGV has been associated with tree decline and graft union necrosis in

sensitive combinations of scion and rootstock in apple and pear (Kundu, 2002).

Citrus tatter leaf virus, a strain of ASGV, causes bud union incompatibility and

necrosis when grafted on sensitive citrus material (Calavan et al., 1963; Miyakawa

and Matsui, 1977; Miyakawa and Ito, 2000; Ito et al., 2003). ASGV is reported to

causes bud-union creases in citrus trees grafted on trifoliate orange rootstocks

(Kusano and Ibi, 2003). In pear ASGV is known to causes Pear black necrotic leaf

spot (PBNLS) disease. Wu et al. (2010) provided conclusive evidence revealing that

ASGV was the causal agent of the pear disease displaying symptoms of reduced size

of foliage and leaf distortion in Taiwan. Virginia Crab stem grooving virus,

Chenopodium dark green epinasty virus and Brown line disease virus are some

synonyms of ASGV.

In susceptible Malus species include severe xylem pitting and grooving with

pegs protruding on innerbark face, phloem necrosis, reduced vigour of the canopy

and an overall decline of the plant. ASGV produces chlorotic leaf spots, stem

grooves and pits, union necrosis and swelling of the stem above the graft union

symptoms on 'Virginia Crab'. Scions and interstocks did not usually show wood

symptoms. Plants grafted with infected material displayed poor budwood welding,

developed poorly, and either died at the nursery or declined in the orchard (Nickel et

al., 2001). In a study by Gong et al. (2002) inoculation by ASGV resulted in poor

growth of the two-year-old pear trees and seedlings due to the sharp decrease of the

three endogenous hormones (indole-3-acetic acid, gibberellic acid and cytokinins)

caused by the infection. Results of studies in 2004 by Maxim et al. on the influence

of ASGV on tree growth of various apple cultivars in the nursery showed that the

virus negatively influenced the tree growth. The average tree height of the 14


35

cultivars infected by ASGV was 23.4% lower and the average diameter 13.7%

smaller than the healthy trees. The most drastic reduction in tree growth were

recorded with 'Golden delicious' cv. where the tree height was by 64.4% lower and

the diameter by 42.9% smaller than the healthy control. The symptom intensity

varied from one cultivar to another and not all the cultivars analyzed showed foliage

symptoms of viral infections.

Similar studies to determine the effects of ASGV on external and physical

characteristic of some commercial apple (Malus dometsica Borkh.) cultivars were

carried out in Turkey during 2006-2008.The results demonstrated that ASGV had no

statistically important effects on tree length, number of the branches, average and

total length of the branches, and leaf dry matter. However, ASGV decreased the

trunk diameter by about 18%, and the woody dry matter in a statistically significant

rate, whereas the angle of the branches from the trunk increased on average about

41% by ASGV infection. The cultivars reacted differently to virus inoculation and

stem grooving symptoms were observed on some tested cultivars (Birişik and

Baloğlu, 2010).

The virus is known to be serologically related to Potato virus T (PVT)

(Salazar and Harrison, 1977; 1978). The virus does not show serological

relationships to ACLSV (Lister et al., 1965; De Sequeira, 1967). The best test for

diagnosis of ASGV is that the virus is not transmitted to Solanum tuberosum, like

PVT (Salazar and Harrison, 1978), nor to Nicotiana glutinosa like ACLSV.

Symptoms in Chenopodium quinoa, Phaseolus vulgaris, Russian apple R-12740-7A

and Virginia Crab also distinguish ACLSV and ASGV. In cross-protection

experiments on Virginia Crab apple the virus seemed to be unrelated to ACLSV (De

Sequeira and Lister, 1969; Bem and Murant, 1979).

Kundu (2003b) performed RT-PCR to determine the occurrence of ASPV

and ASGV in field-grown apple cultivars and found that 44% of the apple cultivars

tested were infected with ASGV. The complete nucleotide sequence of ASGV was

determined by Yoshikawa et al., (1992) and found that it contains ss-RNA and 6495

nt. The results of the molecular characterization of ASGV infecting apple plants in

Santa Catarina, Brazil indicated low coat protein gene variability among Capillovirus

isolates from distinct regions. Two areas of high variability, V1 from amino acid (aa)

530-570 and V2 from aa 1583-1868were identified by Tatineni et al. (2009) in

CTLV. In a restricted survey, mother stocks in orchards and plants introduced into


36

the country for large scale fruit production were indexed and shown to be infected by

ASGV (20%), usually in a complex with other (80%) latent apple viruses (Nickel et

al., 2001).

ASGV is reported to be associated with topworking disease of apple

rootstocks originating from Malus sieboldii (Yanase, 1974; 1981). However, M.

sieboldii and its hybrids confer resistance to apple proliferation (AP) disease. A study

to understand the influence of latent viruses on phytoplasma resistant genotypes was

conducted by Liebenberg et al. (2010). ASGV was successfully maintained in

micropropagated apple trees and was transmitted by in vitro grafting to various

genotypes, for studying in vitro the effect of the virus and virus/phytoplama

combination on M. sieboldii-derived genotypes. M. sieboldii showed a high incidence

of graft union necrosis when grafted with ASGV infected while, no necrosis was

observed on the Golden Delicious controls.

The suitability of different apple tissues for ASGV detection throughout the

year in Czech Republic was checked by RT-PCR and ELISA. Detectable amounts of

ASGV were generally found in all tissues (bark, dormant buds, petals and leaves)

tested by RT-PCR from January to mid-June. Leaves during flowering (in May) were

the most suitable tissues for the virus detection by both methods (RT-PCR and

ELISA). The leaves collected in summer (June, July and August) or other tissues

such as bark, dormant buds and petals were not reliable for ASGV detection by

ELISA (Kundu et al., 2003a).

Hirata et al. (2003) reported that translationally silent nucleotide substitution,

U to C, at nucleotide 4646 within open reading frame (ORF) 1 of ASGV caused

symptom attenuation. Northern and Western blot analyses showed that less ASGV-

RM21 accumulates in host plants than ASGV-wt (wild type). In addition, two more

silent substitutions, U to A and U to G, constructed by site-directed mutagenesis at

the same nucleotide (4646), also induced attenuated symptoms. This was the first

report that a single silent substitution attenuates virus-infection symptoms and

implicates a novel determinant of disease symptom severity. A new strain of ASGV

was identified from Actinidia chinensis (kiwi) imported from China by Clover et al.

(2003).

Nickel et al. (2004) developed polyclonal antibodies to the coat protein of

ASGV expressed in Escherichia coli for use in immunodiagnosis. Shim et al. (2004)

observed capillovirus-like particles under the electron microscope from PBNLS


37

disease infected pear sample. The disease sample was analyzed by western blot and

analyzed with antisera raised against ASGV coat protein. Morphological and

serological analysis, and sequence comparison between the putative pear virus,

ASGV, CTLV and CVA capilloviruses indicated that PBNLS disease may be caused

by a Korean isolate of ASGV.

A magnetic bead-based immunocapture system using polyclonal antiserum

against ASGV successfully facilitated PCR amplification of sequences from three

CTLV isolates originally isolated from the citrus host Meyer lemon (Hilf, 2008).

In India, ASGV was detected by ELISA from apple, plum, cherry, nectarine

and quince (Negi et al., 2009). ASGV coat protein has also been characterized

recently at molecular level from apple for the first time (Negi et al., 2009; 2010).

2.6 APPLE STEM PITTING FOVEAVIRUS

Apple stem pitting virus (ASPV) was reported in 1954 by Smith from USA in M.

sylvestris.

The virus is a member of genus Foveavirus with flexuous filamentous c. 1250 nm

long particles. It has ssRNA genome of 9306 nt (Jelkmann, 1994). The virus was

transmitted by inoculation of sap from stem pitting-diseased apples to Nicotiana

occidentalis and partially characterized by Martelli and Jelkmann (1998). Analysis of

the putative open reading frames (ORFs) showed that it five ORFs in the positive

strand, encoding proteins with 247K (ORF1), 25K (ORF2), 13K (ORF3), 8K (ORF4)

and 42K (ORF5), respectively (Fig 2.6).

Four types of particles with the length of 800, 1600, 2400, and 3200 nm have

been observed in ASPV infected cells. The elongated, flexuous rod shaped particles

of ASPV were found in mesophyll, epidermal and vascular parenchyma cells of

infected plants and are also known to causes cellular changes like disorganization of

chloroplasts. These flexuous particles are reported to have a strong tendency to form

end-to-end aggregates (Koganezawa and Yanase, 1990).

The incidence of the virus infection is usually symptoms less (Németh,

1986a) and occurs together with other pome fruit viruses in mixed infection (Kundu,

2003b; Leone et al., 1998). ASPV is widespread in commercial apple and pear

(Rossini et al., 2010) cultivars which have symptomless infection, unless they are

grafted on sensitive rootstocks. ASPV is known to cause a disease associated with

the tree decline, stem pitting, graft union necrosis and vein yellows in apples and


38

pears (Welsh and Uyemoto, 1980; Desvignes et al., 1990; Kundu, 2002). Like

ASGV, ASPV caused virus symptoms on 'Virginia Crab' such as stem pitting of

xylem that generally stops at the graft union. ASPV also causes fluted (grooved) fruit

in 'Virginia Crab', and epinasty. Depressions and grooving develop on the stem and

the limbs below the union, and longitudinal sutures appear on the bark. In its mild

form, symptoms develop only after the second year following inoculation. Symptoms

consisting of pitting of the woody cylinder, or epinasty and decline, develop in some

ornamental Malus species and in Virigina Crab and Spy 222 indicators. It causes

apple Spy 227 epinasty and decline disease in Spy 222 indicators, while apple dwarf

disease in M. platycarpa. Moreover, a close relationship was reported between

ASPV and the causal agent of necrotic spot and vein yellows diseases of pear

(Lemoine, 1979). Apple spy 227 epinasty and decline virus (Gilmer, 1962; Mink et

al., 1971; Desvignes and Savio, 1975), hawthorn ring pattern mosaic virus, pear

necrotic spot virus (Yanase et al., 1989; Koganezawa and Yanase, 1990), pear stony

pit virus (Koganezawa and Yanase, 1990; Németh, 1986a; Van der Meer, 1986), pear

vein yellows virus (Yanase et al., 1989; Van der Meer, 1986) are few synonyms for

ASPV.

Cropley and Posnette (1973) studied the effect of viruses on growth and

cropping of pear trees for 13 years. They reported that, vein yellows and mosaic

reduced the growth and crop of 4 cultivars (Beurre Hardy, Doyenne du Comice,

Conference and Williams' Bon Chretien). Reduction in yield was mainly due to the

smaller size of infected trees. These produced about 30% less fruit than healthy trees

when infected with vein yellows, and 40% less fruit when also infected with mosaic.

Fruit size was decreased in only one year. The fertility of Doyenne du Comice

flowers was also observed to be impaired. Klerks et al. (2001) have developed a

rapid and sensitive gel free detection system for ASPV in apple trees through RNA

amplification and probing with fluorescent molecular beacons. Yoshikawa et al.

(2001) analyzed genome heterogeneity of ASPV in apple trees. The complete

nucleotide sequence of ASPV isolate IF38 was also achieved.

Mathioudakis et al. (2006) recorded symptoms of Quince Fruit Deformation

Disease (QFDD) in a quince orchard in northwestern Greece. Direct sequencing of

one PCR product PCR using a pair of degenerate primers confirmed the specific

detection of ASPV. Sequence analysis of coat protein (CP) gene from 6 isolates of

ASPV showed nucleotide identities ranged from 68.7 to 99.7%. Phylogenetic


39

analysis showed that all ASPV isolates fell into three groups. To a certain extent, the

groups were related to the host origins of virus. The hypervariable and conserved

region could be found in the CP gene. The variations of nucleotide sequence and

predicted RNA secondary structure were analyzed for the CP gene, and the

characteristic of RNA secondary structure of ASPV 5' and 3' untranslated region

(UTR) were also studied. The result indicated that predicted RNA secondary

structures could play an important role in studying the molecular variation of virus.

The 3' UTR of ASPV RNA was predicted to contain four stem-loop structures, and it

was likely to imply that the variation of RNA secondary structure was related to

groups of phylogenetic analysis (Li et al., 2009). A study of the distribution of ASPV

in tissues of pear tree using in situ hybridization and in situ RT-PCR was done by

Zhao et al. (2009). ASPV was found to be mainly distributed in palisade tissue of

mesophyll cells, external cortex of the tip, and the corresponding newborn vascular

bundles. The suitable annealing temperature in PCR reaction was 60°C with 35

cycles. The apical meristem region of 0.25 mm was found to be virus-free.

Evaluation of ASPV detection using RT-PCR based methods was studied in

infected apple and pear trees. Three virus-specific primers were designed to target

the most conservative regions of the coat protein gene for the experiment. Besides,

the comparative analysis of silicacapture-RT-PCR (SC-RT-PCR) versus

immunocapture-RT-PCR (IC-RT-PCR) assays was also carried out. Few ASPV

isolates escaped detection by IC-RT-PCR, while all isolates tested were detected

using the SC-RT-PCR with the new primers (Komorowska et al., 2010). Recently,

ASPV has also been molecularly characterized from India by Dhir et al. (2010)

2.7 MINOR VIRUSES

Minor viruses do not cause major economic loss to the yield however, apple and

other pome and stone fruits serve as natural alternative hosts to these. Some of the

viruses are:

i. Prunus necrotic ringspot virus (PNRSV)

ii. Cherry rasp leaf virus (CRLV)

iii. Little cherry virus (LChV)

iv. Tobacco ringspot virus (TRSV)

v. Tomato ringspot virus (ToRSV)

vi. Tomato black ring virus (TBRV)


40

vii. Arabis mosaic virus (ArMV)

viii. Apple latent spherical virus (ALSV)

ix. Grapevine berry inner necrosis virus (GINV)

2.7.1 Family-Bromoviridae Genus-Ilarvirus

2.7.1.1 Prunus necrotic ringspot virus

Prunus necrotic ringspot virus (PNRSV), a definitive Ilarvirus species assigned to

subgroup III (Roossinck et al., 2000), was first described as Peach ringspot virus

from peach plants showing severe ringspots (Cochran and Hutchins, 1941). PNRSV

posses a tripartite genome and morphologically isometric (25-30 run) to bacilliform

shaped particles. The virus was common and widespread in different stone fruits,

causing crop losses (Barbara, 1988; Mink, 1992). PNRSV has been reported to be

phylogenetically related to Prune dwarf virus (PDV) and ApMV (Sánchez-Navarro

and Pallás, 1997). Serologically, PNRSV is distantly related to ApMV (Fulton,

1968), which is included in the same subgroup III (Fulton, 1968; Casper, 1973; Halk

et al., 1984; Sek-Man and Kenneth-Horst, 1993; Roossinck et al., 2000). The coat

protein and movement protein of these two viruses are sufficiently conserved so as to

suggest that they have evolved from a common ancestor (Scott et al., 1998). PNRSV

was also shown to be molecularly close to Alfalfa mosaic virus, especially in the

nucleotide sequence of the coat and movement proteins (Sánchez-Navarro and

Pallás, 1997).

PNRSV can be transmitted by grafting and persists in propagative material

(budwood, rootstocks and grafted nursery plants) of all host species. The virus is also

pollen-borne being carried externally and internally on pollen grains (Cole et al.,

1982; Digiaro and Savino, 1992; Aparicio et al., 1999). Infected pollen is regarded as

the major agent of virus spread which, in nature, is mediated by pollinating insects,

especially honeybees (George and Davidson, 1963; Davidson, 1976). Economically

PNRSV is a major virus in stone fruits causing severe reduction in peach production

by decreasing the number of flower buds and the quality and quantity of the yield

with losses of up to 95% of the crop (Schmitt et al., 1981; Lazarova-Topchiiska,

1984; Wood et al., 1997). Growth and yield reduction up to 60% was observed in

sour and sweet cherries (Hilsendengen, 1999), and average losses of 25% were

recorded from almond affected by almond mosaic, a complex disease in which

PNRSV is involved (Martelli and Savino, 1997). On apple plants however, PNRSV


41

is a minor virus. Nicolaescu reported that pollen from PNRSV-infected apple trees

showed a much lower germination percentage and pollen tube length was also

greatly reduced (1972).

According to the virus strain, PNRSV infections can vary from very severe,

to mild, to symptomless, and can visibly affect leaves, shoots and fruits (Fulton,

1981; Németh, 1986a; Desvignes, 1999). The initial symptomatic shock phase is

usually followed by symptomless chronic infection. Symptoms are characterized by

discoloration or localized necrosis of foliar tissues, flowers (colour breaking of peach

flowers), buds (bud failure of almond, peach mule's ear), and shoots (terminal die-

back). Discoloration in the form of chlorotic or bright yellow blotching (almond

calico), chlorotic to yellow mottling, ringspots, line and oak leaf patterns, diffuse

yellowing (cherry dusty yellows), are especially conspicuous in spring on newly

developed vegetation, but tend to fade as the season advances.

Localized necrotic reactions (shock symptoms) can develop on young leaves,

resulting in perforations and shredding of the blades which, if the leaves are not shed,

remain visible throughout the vegetative season (cherry tatter leaf, cherry

Stecklenberger disease, apricot necrotic ringspot, peach necrotic leaf spot, peach

tatter leaf). Some virus strains induce enations and severe deformation of the leaves

(cherry rugose mosaic, cherry lace leaf). PNRSV infection can be detected in all

hosts by visual examination in the field, especially in spring, during the shock phase.

Field detection during chronic infection may be difficult or impossible (Fulton, 1981;

Desvignes, 1999). Infected trees are usually distributed at random in the orchards.

More recent comparison of three sensitive detection techniques, i.e. double antibody

sandwich (DAS)-ELISA, non isotopic dot blot molecular hybridization, and RT-

PCR, showed that the detection limit of non isotopic hybridization was 25 times

higher than that of DAS-ELISA and 625 times lower than that of RT-PCR (Sánchez-

Navarro et al., 1998). Zotto et al. (1999) researched the fluctuations in PNRSV

concentration in single plants of six peach (Prunus persicae) cultivars- Kurakata,

Red Haven, Nectar Red, Start Delicious, Meadowlark, and Loadel by double

antibody sandwich–enzyme-linked immunosorbent assay (DAS-ELISA) of dormant

buds (May, June), flowers (September), new sprouts (November), and mature leaves

(January) (in Southern Hemisphere). It was concluded that concentration of PNRSV

varies seasonally in peach trees, although fluctuations are not identical in all

cultivars. In general, virus from dormant buds of all cultivars was reliable for


42

distinguishing between healthy and infected plants, while flowers and new sprouts

gave high values in some cultivars but not in others.

The laboratory studies by Milne and Walter (2003) have demonstrated that PNRSV

(family Bromoviridae) can be readily transmitted when thrips and virus-bearing

pollen are placed together on to test plants. The results indicated that the common

thrips species present on stone fruit trees in the Granite Belt were also ones

previously shown to transmit PNRSV via infected pollen in the laboratory and that

these thrips were concentrated in stone fruit flowers where most stone fruit pollen

was deposited. These results contributed to mounting circumstantial evidence that

stone fruit flowers may be inoculated with PNRSV via an interaction of thrips with

virus-bearing pollen and that this transmission mechanism may be an important

cause of new tree infections in the field.

Maliogka et al. (2007) developed nested PCR assays for the generic detection

of ilarviruses amplifying a 371 bp RdRp fragment. Later in 2010 they used the

nested PCR assays for simultaneous detection of PDV, PNRSV and ApMV in

almond and cherry plantation in Greece.

Simultaneous infections of peach (Prunus persica Batsch L.) with the two

ilarviruses, PNRSV and PDV, produce a synergistic disease referred to as “peach

stunt”. Experiments by Scott et al. (2001) showed significant differences in the

expression of the coat protein (CP) genes. In the presence of PNRSV, an up to 17-

fold reduction in the amount of (+) strand RNA 3 of PDV, as compared to similar

trees infected with PDV alone, was observed. However, the presence of PDV had no

effect on the concentration of (+) strands of RNA 3 of PNRSV. Repetition of the

same experiment by Kim et al. (2010) confirms delay in the replication

/accumulation of PDV in buds at the beginning though, reduction in the amount of

(+) strand RNA 3 of PDV was not achieved.

Aparicio et al. (2010) studied the implication of the C terminus of the

PNRSV movement protein (MP) in cell-to-cell transport and in its interaction with

the coat protein. Using biomolecular fluorescence complementation and overlay

analysis interaction between the C-terminal 38 amino acids (aa) of PNRSV MP and

its cognate CP was confirmed. Mutational analysis of the C-terminal region of the

PNRSV MP revealed that its C-terminal 38 aa were dispensable for virus transport

however, the 4 aa preceding the dispensable C-terminus were necessary to target

the

MP to the plasmodesmata and for the


43

functionality of the protein.

From India PNRSV has been reported on peach (Chandel et al., 2007a),

nectarine (Chandel et al., 2008a), wild Himalayan cherry (Chandel et al., 2007b).

PNRSV has also been reported on apple (Chandel et al., 2008b), cherry and almond

on the basis of serological and molecular techniques. Phylogenetic analysis showed

that Indian isolates of PNRSV-CP fall in Group-I (Chandel et al., 2009).

2.7.2 Family-Comoviridae Genus -Nepovirus

The name ‘Nepo’ is derived from nematode, polyhedral particles to distinguish these

viruses from the tobraviruses.

2.7.2.1 Tobacco ringspot virus

Tobacco ringspot virus (TRSV) is the type species of genus Nepovirus. The genome

is segmented, bipartite, segements are distributed among 2 particle types of linear,

positive-sense, single-stranded RNA. The encapsidated nucleic acid is mainly of

genomic origin, but virions may also contain satellite RNA (in some strains). The

complete genome is 13600 nucleotides long. TRSV is reported to cause ringspot

diseases of tobacco, cucumber, Easter lily, hydrangea, iris and Pelargonium; also

blueberry necrotic ringspot, soybean bud blight, and chlorotic or necrotic spotting in

many other annual and perennial crops. It is transmitted by the nematode Xiphinema

americanum (McGuire, 1964), and also by species of thrips, spider mite,

grasshopper, flea beetle and, possibly, aphid. It is commonly seed transmitted.

TRSV infects cherry (sweet and ornamental) naturally (Uyemoto et al.,

1977). It is known to cause cholorotic blotching over the leaf blade in young leaves,

deformed leaf margins, lobed and late maturing fruits on infected tree. Stace-Smith

reported TRSV to cause delay in bud opening and flowering and irregular light-green

leaf blotches (1985). TRSV is a minor virus in apple and other pome fruits. Allan

Femi Lana et al. (2008) reported association of union incompatibility condition of

apple and TRSV. Apple union necrosis and decline was associated with the recovery

of TRSV from infected rootstocks in Canada. In India, sporadic occurrence of TRSV

in pome and stone fruits has been observed from time to time using ELISA

(Noorani et al. unpublished).


44

2.7.2.2 Tomato ringspot virus

The Tomato ringspot virus (ToRSV) is a distinctive member of the Nepovirus group

and is unrelated serologically to any other member. Stace-Smith and Ramsdell

(1987) reported economically important disease in stone fruits in Eastern and western

USA caused by ToRSV. The virus occurs in nature mostly in perennial crops. The

virus is irregularly distributed in some hosts, often samples from lower trunk are

most reliable (Bitterlin and Gonsalves, 1986; Powell et al., 1991). Strains of ToRSV

have been isolated from peach, grape, tobacco and apple (Stace-Smith 1996).

The most serious diseases are yellow bud mosaic in peach and other Prunus

spp. (Schlocker and Traylor, 1976), stem pitting and decline in peach, cherry,

apricot, plum and other Prunus spp. (Mircetich and Civerolo, 1972; Smith et al.,

1973; Mircetich and Fogle, 1976), ‘brownline’ disease of prune (Hoy et al., 1984),

union necrosis and decline in apple nurseries (Stouffer et al., 1977), ringspot and

decline in red raspberry (Stace-Smith, 1984) and decline in grapevine (Allen and van

Schagen, 1982). Most infected plants showed distinctive symptoms as a shock

reaction; chronically infected plants usually exhibited no obvious symptoms but

showed a general decline in productivity. It caused symptoms like stem pitting and

decline in peach, cherry, apricot and plum. Other symptoms included reduced

terminal growth, chlorotic leaves curling upwards and turning red in autumn,

premature defoliation, enlargement of lower trunk with very thick spongy bark and

necrotic areas. The virus moves slowly upwards so that, after several years, fruit is

produced only at the branch extremities. Symptoms may vary depending on the age

of the tree or the duration of the infection and on climate changes from year to year

(Schlocker and Traylor, 1976). Forer et al. (1981) reported transmission of ToRSV

to apple rootstock cuttings by Xiphinema rivesi.

Salem et al. (2005) conducted surveys in the traditional areas of Jordan to

assess the phytosanitary status of apple, pear, and quince species. Samples were

checked using DAS-ELISA for ACLSV, ApMV, ASGV and ToRSV. All four

viruses were identified in a large number of these samples, ToRSV was the most

widespread and ASGV was the second most prevalent. Youssef and Shalaby (2009)

developed one single-step multiplex reverse transcription-polymerase chain reaction

(m-RT-PCR) by designing five compatible primer sets for the simultaneous detection

and discrimination among five RNA viruses namely ACLSV, PDV, PNRSV, PPV

and ToRSV. A field surveys was conducted in Jordan to assess the incidence of


45

ToRSV infection in stone fruit trees by Al-Nsour et al. (2010). The disease incidence

percentages based on DAS-ELISA for apricot, almond, peach, nectarine, plum and

cherry trees were 10, 14, 15, 19, 22 and 28% of the tested trees, respectively. The In

Iran apple (Malus domestica) showing vein yellowing was identified as a natural host

of ToRSV by Moini (2010). Mechanical inoculation of enzyme-linked

immunosorbent assay (ELISA)-positive samples on Chenopodium spp. and

Nicotiana tabacum induced local lesions and systemic infection, respectively.

Transmission from the ToRSV-infected tobacco onto apple seedlings resulted in

systemic infection and vein yellowing with positive reaction in ELISA.

2.7.2.3 Tomato black ring virus

The Tomato black ring virus (TBRV) belongs to Nepovirus group and symptoms are

characterized by leaves having irregular chlorotic spots and distorted laminae. It has

been detected sporadically in peach, sweet cherry, and almond trees (Németh,

1986a). The hosts also include important berry and fruit plants (Rubus, Ribes,

Fragaria and Prunus), sugar beet, potatoes and different vegetables (Allium,

Brassica, Solanum and Phaseolus) (Andersson, 2010). The genome of nepoviruses

consists of two RNAs: RNA1 has genes for replication and protein processing and

RNA2 has genes for the coat protein (CP) and virus movement. The CP gene is very

variable and is usually species specific, which makes it suitable for identifying and

distinguishing virus species (Le Gall et al., 1995). Nepoviruses are divided into three

subgroups; a, b and c depending on the size of the RNA2 (Steinkellner et al. 1992).

TBRV has been shown to have a wide host range and has spread all over the

world. It is also not an economically important virus and is of concern only in

nurseries. Infected crop plants do not usually develop diagnostic symptoms. TRBV is

transmitted by a large portion of seeds of many host plants and weeds (Murant, 1987)

and also by nematodes Logidorous attenuates and L. elungatus (Murant 1970).

TBRV is readily transmitted by inoculation of sap to many herbaceous test plants but

mechanical inoculation of virus from woody plants should be made in 2% (v/v)

nicotine sulfate (pH 9.3). In test species such as Chenopodium quinoa, C.

amaranticolor and Nicotiana clevelandii, TBRV induced chlorotic or necrotic local

lesions and systemic necrosis, depending on the virus isolate. To establish

unequivocally the presence of TBRV serological tests were generally considered the

most convenient while, ELISA as probably the most sensitive. However, because of


46

the serological variability of TBRV isolates, antisera to each of the two main

serotype groups should be used (Bercks, 1963). ELISA has been successfully used to

detect TBRV directly in plants such as grapevine, raspberry and strawberry. Gene

sequences of the viral genome of components RNA1 and RNA2 were elucidated by

Jończyk et al. (2004a). The genetic variability of the virus isolates infecting

vegetable crops was also established (Jończyk et al., 2004b).

2.7.2.3 Arabis mosaic virus

Arabis mosaic virus (ArMV) is a member of Comoviridae, genus Nepovirus. ArMV

has a bipartite genome with two RNA-species. It is transmitted by its nematode

vector over short distances only. Principal hosts are strawberries, hops, Vitis spp.,

raspberries (Rubus idaeus), Rheum spp. and Sambucus nigra (EPPO, 1996). Till date

it has not been detected on apple. Diseases caused by ArMV are generally of a local

and/or crop-specific character but can have a devastating effect where they occur.

Strawberries and raspberries can be severely affected and in some cultivars plants

may even be killed by the virus. The diseases caused in certain cultivars are called

mosaic and yellow crinkle of strawberry and yellow dwarf of raspberry. In cherries,

a mixed infection of ArMV with PDV or PNRSV is reported to induce 'European

rasp leaf' (EPPO, 1996).

2.7.3 Family- Secoviridae Genus -Cheravirus

The name ‘Chera’ is derived from Cherry, its natural host and rasp leaf symptoms

characteristic to its type species.

2.7.3.1 Cherry rasp leaf virus

Rasp leaf is a disease of cherry, first reported from Colorado (Bodine and Newton,

1942). Cherry rasp leaf virus (CRLV) previously considered as atypical but tentative

members of Nepovirus genus (family Comoviridae) was proposed as a type species

of new genus Cheravirus in newly formed family Secoviridae (Le Gall et al., 2005).

The isometric virus particles are polyhedral in outline. The genome is segmented;

bipartite, segements are distributed among 2 particle types of linear, positive-sense,

single-stranded RNA. The complete genome is 13300 nucleotides long. The

multipartite genome is divided among different particles, each segment encapsidated

separately and the segments are distributed between 2 different types of particles.


47

The smallest particles contain no nucleic acid and were found in the top (sedimenting

component T) band after sedimentation (Stace-Smith and Hansen, 1976a, b).

Virus is transmitted by nematodes, family Dorylamidae- Xiphinema

americanum (Nyland et al., 1969; Hansen et al., 1974) and can be best detected in

parenchyma, plasmodesmata (Stace- Smith and Hansen, 1976b) and cytoplasm.

Virus is also reported to be transmitted by grafting, seeds (10-20%) in some weed

species and mechanical inoculation to a wide range of herbaceous hosts

(OEPP/EPPO, 1984). Typical of other members of this genus, it has a wide host

range of herbaceous and woody plants. The characteristic disease symptoms are

prominent enations on the underside of the leaves (Ogawa et al., 1991a). In apple

(Malus sylvestris), the virus is incitant of ‘flat apple' disease (Parish, 1977; Hansen

and Parish, 1990; James and Upton, 2005). The taste and appearance of the fruit are

affected, reducing their value or even making them unmarketable (Németh, 1986a).

Symptoms of flat apple as described on cvs. Red Delicious and Golden Delicious

include small flattened fruit, reduced lateral branch growth, and upward rolling

leaves (Hansen and Parish, 1990). Cherry rasp leaf disease in cherry reduces fruit

production, tree vigor and life expectancy (Nyland, 1974). It also infects several

weeds such as dandelion (Park and Kim, 2004). CRLV can cause serious stunting of

infected peach (Stace-Smith and Hansen, 1976a) trees, and fruit yield and quality

reductions in both cherries and apples. In addition, young trees and seedling

rootstocks are sometimes killed. In older orchards, CRLV can reach high levels of

infection, and trees planted on previously infected sites can also become infected.

Symptomless infection has also been reported in raspberry (Rubus idaeus) (Jones et

al., 1985).

CRLV is unrelated serologically to definite species of the Nepovirus genus,

such as TRSV, ToRSV, Cherry leaf roll virus (CLRV), Peach rosette mosaic virus,

Raspberry ringspot virus, Strawberry latent ringspot virus, TBRV and ArMV

(Stace-Smith and Hansen, 1976a; Hansen, 1995b). Available molecular data indicate

that CRLV is more closely related to Apple latent spherical virus (ALSV) than to

other members of the family Comoviridae (James et al., 2001). ALSV is a new

member of this family with no known vector. It has been proposed that ALSV be

classified in a new genus. ALSV and CRLV are not related serologically (Li et al.,

2000).


48

Oligonucleotide primers have been developed that allow reliable RT-PCR

detection of CRLV in Chenopodium quinoa, cherry and apple (James et al., 2001).

The virus was reliably detected in leaf and budwood tissue of cherry and apple. The

sequence of RNA-2 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) was

determined and consisted of 3274 nucleotides, excluding a 3' poly (A) tail. The data

supports re-classification of CRLV in a new genus in the family Comoviridae. A

single open reading frame (ORF) encoding a putative 108 kDa polyprotein was

identified. Potential protease cleavage sites were identified which would result in the

production of a putative movement protein (41 kDa), and 3 capsid protein subunits

(24, 20, and 22 kDa, respectively). A 5'-UTR and 3'-UTR were identified, 248 nt and

146 nt long respectively. The genome organisation of CRLV-FA RNA-2 is similar to

that of Apple latent spherical virus (ALSV) RNA-2, a new member of the family

Comoviridae. The Vp25 amino acid sequences were unique to CRLV-FA and ALSV

(54% identity), with no relationship identified to any other virus. CRLV-FA Vp20

and Vp24 amino acid sequences were closely related to ALSV (59 and 65%,

respectively) but the only other relationships identified were with a range of animal

ssRNA positive-strand viruses (James and Upton, 2002). The sequence of the RNA-1

of CRLV-F was also obtained using overlapping cDNA fragments. CRLV-FA RNA-

1 consisted of 6992 nucleotides (nt), excluding a 3' poly (A) tail. A single open

reading frame (ORF) consisting of 6705 nt was identified. This ORF encodes a

putative polyprotein consisting of 2235 amino acid (aa) residues, approximately

249.6 kDa (James and Upton, 2005).

In the Indian scenario, characteristic symptoms of enations on leaf have been

observed on cherry from Kashmir and flat apple like symptoms of fruit have been

reported from time to time in HP and Kashmir. Till date CRLV has been detected

from apple and cherry by ELISA and tissue slot blot hybridization using CRLV

probe indicating towards its presence (Noorani et al. unpublished).

2.7.3.2 Apple latent spherical virus

Apple latent spherical virus (ALSV) is classified as a new species in the genus

Cheravirus (Ikegami et al., 2002). Earlier, Li et al. (2000) on basis of phylogenetic

analysis of the RNA polymerase domain showed that the virus should be classified

into the family Sequiviridae, rather than Comoviridae. Virus particles are isometric c.

25nm in diameter, and contain two ssRNA species (RNA1 and RNA2) and three


49

capsid proteins (Vp25, Vp20 and Vp24) (Li et al., 2000). The virus is

phylogenetically related to CRLV-FA (James and Upton, 2002; Thompson et al.,

2004), though ALSV does not induce any obvious disease in apple trees.

Infectious cDNA clones of ALSV-RNA1 and -RNA2 containing Cauliflower

mosaic virus 35S promoter and then engineered an infectious cDNA clone of ALSV-

RNA2 into expression vectors. The ALSV-RNA2 vectors could be used for stable

expression of foreign genes in plants (Li et al., 2004). It was demonstrated that for

the ALSV vector which expresses GFP (GFP-ALSV), used for the tracing of cell-to-

cell movement of ALSV in infected tissues, the MP and the three capsid proteins are

all indispensable for the cell-to-cell movement of the virus (Yoshikawa et al.,

2006a). ALSV was reported to efficiently induce VIGS in infected plants because of

a lack of silencing suppressor strong enough to suppress local silencing. It might

have evolved mechanisms to evade RNA silencing instead of the suppression of local

silencing for the establishment of systemic infection e.g. rapid encapsidation of a

virus and encoding a suppressor to interfere with systemic silencing. Vp20 encoded

by ALSV-RNA2 was reported to have suppressor activity to interfere with systemic

silencing (Yaegashi et al., 2007b).

Recently, ALSV-based vectors were used for reliable and effective VIGS and

expression of foreign proteins in a broad range of plants (Li et al. 2004; Igarashi et

al. 2009). However, the infection efficiency of ALSV inoculation to apple trees by

conventional methods was reported to be poor (Ito et al., 1992; Li et al., 2004).

Experiments by Yamagishi et al. (2010) showed that biolistic inoculation in

germinated apple seeds was efficient inoculation method of apple viruses. The

method achieved high and reproducible infection efficiency and could be applied to

other virus-fruit tree combinations to satisfy Koch’s postulates, to test resistance to

viruses in breeding programs, and to analyze gene function by virus vectors etc.

2.7.4 Family- Closteroviridae

2.7.4.1 Little cherry virus (Genus-Closterovirus)

Little cherry disease has been associated with two different long flexuous

filamentous viruses of the family Closteroviridae. Little cherry virus -1 (LChV-1) is


50

an unassigned member in the family while Little cherry virus -2 (LChV-2) has been

assigned to the genus Ampelovirus. Both viruses have been characterized at the

molecular level (Rott and Jelkmann, 2005; Jelkmann et al., 1997). The virus is quite

widespread in various parts of the world including Europe, US, Japan, Canada and

New Zealand.

Antisera for LChV-1 produced from purified virus preparations, bacterially

expressed protein, or a DNA vector that expressed the cloned coat protein (CP) gene

in vivo have been described (Matic et al. 2009a; KeimKonrad and Jelkmann 1996).

Symptoms are characterized by leaves showing reddening and bronzing, black line,

leaf rolling and delay in flowering followed by plant death. Fruits from the infected

trees are initially normal but fail to fully ripen. Fruits are poorly coloured, small and

insipid in taste. The virus has been reported on cherry and peach (Prunus persica)

(Brunt, 1966; Jones, 1985; Jelkmann et al., 1997; Raine et al., 1975; Eppler et al.,

2001). In addition to cherry, LChV-1 was identified in plum and almond (Matic et al.

2009b). The virus (LChV2) has also been reported on apple (Jelkmann et al., 1997;

Raine et al., 1975; Isogai et al., 2004; Eppler et al., 2001).

LChV-1 and LChV-2 were also detected both alone and in combination in

five sweet cherry orchards for the first time in Washington State. Two sets of primers

corresponding to a portion of the replicase gene of LChV-1 and LChV-2 were used

in one-tube reverse-transcription polymerase chain reactions to detect these viruses

in total RNA extracts of field-collected sweet cherry tissues (Bajet et al., 2008). The

disease is readily graft-transmissible from cherry to cherry. There is no known vector

associated with LChV-1 however, LChV-2 is transmitted by the apple mealybug

(Phenacoccus aceris). Both viruses can be detected by RT-PCR and woody indexing

on sensitive indicator plants (Jelkmann and Eastwell 2010). Recently Jelkmann et al.

(2010) reported transmission of LChV-1 was from infected P. avium F12 rootstocks

by Cuscuta europea to Nicotiana occidentalis ‘37B’. Transmission of the virus was

confirmed by RT-PCR analysis of total nucleic acid extracts from dodder and N.

occidentalis. Symptoms consisted of curled leaves, reddening of leaf margins and

veins, and plant decline. In parallel attempts virus transmission was not successful

for LChV-2. Propagation of LChV-1 by mechanical transmission on N. occidentalis

failed, however the virus was transferred serially by grafting.


51

In India characteristic symptoms of leaf reddening and bronzing and

production of pointed and non uniformly ripened cherry has been observed in the

Kashmir plantations during recent surveys (Noorani et al. unpublished).

2.7.5 Family- Flexiviridae

2.7.5.1 Cherry necrotic rusty mottle virus (Genus-Foveavirus)

Cherry necrotic rusty mottle virus (CNRMV) are found as common pathogens in

commercial apple and cherry cultivars causing significant yield losses and thus

economically affecting farm income (Desvignes et al., 1999; James and Upton, 2002;

Stouffer and Fridund, 1989). CNRMV is a serious disease of sweet cherry in North

America, Europe and New Zealand (Wadley and Lipman, 1976). The most important

disease characteristics are brown angular necrotic spots, rusty chlorotic areas, shot

holes of the leaves, blisters, gum pockets and general necrosis of the bark. The

disease can be transmitted by grafting but not by mechanically (Rott and Jelkmann,

2001).

CNRMV has a genome of 8,432 nucleotides excluding the 3’ poly(A)

sequence and codes for 7 significant open reading frames (ORFs). Five of these

ORFs are conserved among all fovea-, allexi-, potex- and carlaviruses. These ORFs

code for a methyltransferase/helicase/polymerase polypeptide, the triple gene block

movement proteins and the coat protein. Two further ORFs, ORFs 2a and 5a, are

nested completely within ORFs 2 and 5, respectively (Rott and Jelkmann, 2001).

A simple and reliable procedure for simultaneous RT-PCR detection of

ASPV, CRLV and CNRMV was developed by Park and Kim (2004). Li and Mock

(2008) characterized genomic sequences (two isolates) of flowering cherry strain of

CNRMV. The CNRMV-FC4 and CNRMV-FC5 isolates were 8,430 and 8,429 nt in

length, excluding the 3' poly (A) tail. They contained seven open reading frames

encoding for a putative virus replicase, "triple gene block" proteins, a coat protein

and two proteins with unknown functions. The two CNRMV-FC isolates share 96%

identity in the genomic sequences, and their genome organizations are virtually

identical to that of a German CNRMV isolate (CNRMV-GER).

2.7.5.2 Grapevine berry inner necrosis virus (Genus-Trichovirus)

Grapevine berry inner necrosis virus (GINV) belongs in the genus Trichovirus. Host

range and particle length of GINV were quite similar to those of ACLSV.


52

Serological relationships were not found between GINV, ACLSV (Klerks et al.,

2001), grapevine virus A or grapevine virus B (Yoshikawa et al., 1997). GINV is the

agent of the homonymous disease of some Japanese grape varieties (Goszczynski

and Jooste, 2003). GINV has not been reported from apple. However, experiments

with N. occidentalis plants expressing the MP (50K) ACLSV, disclosed resistance to

GINV, a heterologous virus member of the same genus (Yoshikawa et al., 2000).

Severity of symptoms and accumulation of the homologous virus were greatly

enhanced, while specific resistance of transgenic plants to GINV was shown to

depend on the ability of the 50K protein to block intra- and intercellular trafficking

(Isogai et al., 2003)

2.8 VIROID DISEASES

Viroids have a small, GC-rich genome (246–401nucleotides), which does not code

for any proteins. On the basis of biochemical, biological and structural properties,

viroids have been classified into two families, Pospiviroidae and Avsunviroidae,

members of which replicate in nucleus and chloroplast, respectively (Flores et al.,

2005). Twenty-five different viroid sequences have been determined and numerous

variants identified. Pospiviroidae and Avsunviroidae are two families of viroids.

Viroids on apple are common plant pathogens which are a serious economic problem

worldwide because they produce severe symptoms on apple fruits rendering it

unmarketable.

Apple scar skin viroid (ASSVd), Pear blister canker viroid (PBCVd), Apple

fruit crinkle viroid and Apple dimple fruit viroid (ADFVd) (Koganezawa et al., 1982;

Di Serio et al., 2002; Koganezawa et al., 2003) are widely distributed and

economically important pome fruit viroids, all belonging to genus Apscaviroid of

Pospiviroidae family (Faggioli and Ragozzino, 2002). In apple, disease symptoms

most commonly observed are blistering of bark in delicious cultivars and dimpling,

scarring, crinkling, dappling/bumpiness of apple fruits have been characteristic of

viroid infection. These are considered to be serious pathogens in most of the apple

producing countries of the world. The symptoms appear only on the fruit. Scarring or

dapple symptoms appear depending on the cultivars and the viroid variant infecting

apple plantations. The entire crop from affected trees becomes unmarketable and

because apple trees are perennial, the economic loss to apple growers is substantial.


53

Since the pathogen lacks protein coat, its detection is largely carried out by Reverse-

PAGE, NASH and PCR based detection techniques.

2.8.1 Apple scar skin viroid

The apple scar skin disease was first reported in 1938 in China (Manchuria) with the

name “Manshu-sabika-byo” by Ohtsuka (1938). The causal agent i.e., viroid was first

reported by Desvignes et al. (1999). Among important viroids in the family

Pospiviroidae, genus Apscaviroid, Apple scar skin viroid (ASSVd), the type member

of this genus, was the first viroid reported to infect pome fruit trees of Malus, Pyrus

and Cydonia spp. (Hashimoto and Koganezawa, 1987). It has been reported in apple

(Malus domestica), pear (Pyrus communis, P. pyrifolia), wild apple (M. sylvestris)

and wild pear (P. amygdaliformis) (Kyriakopoulou and Hadidi 1998; Kyriakopoulou

et al., 2001; 2003; Koganezawa et al., 2003; Boubourakas et al., 2008). Recently, it

was reported in Chinese peach, apricot (Zhao and Niu, 2006; 2008) and sweet cherry

(Kaponi et al., 2010).

Studies conducted over the last 10 years have revealed that the disease caused

by ASSVd was extremely rare in Europe. There was little or no seed transmission of

this viroid. However, ASSVd was transmitted at a low rate under field conditions to

adjacent trees (Desvignes et al., 1999). However, two apple disorders were described

simultaneously in the U.S.A: the apple scar skin disease on cv. Red Delicious in

Missouri and the dapple apple disease on cv. Cortland. The two diseases were

attributed to the same infectious agent that was quite widespread on apple. This

infectious agent was identified to be a viroid, called ASSVd consisting of 330

nucleotides. ASSVd has also been reported from several parts of Asia like Japan

(Hashimoto and Koganezawa, 1987), China (Puchta et al., 1990) and Korea.

Mutational analysis of Potato spindle tuber viroid (PSTVd) , the most worked

member of family Pospiviroidae, and construction of intra-specific chimeras have

shown that sequences within at least three of these domains (i.e. the left terminal

loop, pathogenecity domain and variable domain/right terminal loop) had important

role in modulating symptom expression (Visvader and Symons, 1986; Sano et al.,

1992). ASSVd caused symptoms of dappling (in new apple cultivars) as well as

scarring (in old apple cultivars). The ASSVd symptoms were observed to be cultivar

dependent (Desvignes et al., 1999) and probably on the viroid variant infecting the

cultivar. A one tube-one step RT-PCR was developed for the detection of seven


54

viroids (ASSVd, ADFVd, Pear blister canker viroid, Hop stunt viroid,

Chrysanthemum stunt viroid, Citrus exocortis viroid and Peach latent mosaic viroid)

in four genera that infect eight plant species (Ragozzino et al., 2004).

In India ASSVd has already been reported based on symptomatology and

PAGE assays (Handa et al., 1998). Recently, ASSVd was confirmed from trees with

symptomatic fruits and molecularly characterized from apple bark tissue from

northern India. Apple plants were found to be infected with complex mixture of

sequence variants of ASSVd. This was the first molecular report of a viroid infection

in apple from India (Walia et al., 2009). Sequence analysis of variants indicated

maximum variability in the pathogenic domain of the viroid genome. Infectious

clone of ASSVd were also constructed for in vitro mutagenic studies (Walia et al.,

2010).

2.8.2 Apple dimple fruit viroid

The Apple dimple fruit viroid (ADFVd), a member of Pospiviroidae, has been

detected in apple extracts by dot blot hybridization using radioactive probe has also

been reported by Skrzeczkowski et al. (1993). ADFVd was a new member of the

ASSVd group whose central and left terminal domains were also present in other

members of this group, whereas the sequences forming its right terminal domain are

more similar to those of some viroids outside the ASSVd group. This observation

suggests that ADFVd could have evolved from recombination events between

viroids of different groups, a situation which appears to be frequent in members of

the ASSVd group (Di Serio et al., 1996). More recently, pome-fruit viroids have

been detected by RT-PCR using single primer pair (Faggioli and Ragozzino, 2002).

ADFVd was found to be 306 nucleotides long. Initial symptoms appeared six weeks

after pollination on the stem end of affected fruits as water-soaked blotches. Similar

but smaller spots appeared on the sides as well. Two to three weeks later, scared

tissue developed around the calyx end, later scar patches appear on the skin giving

the fruits a corky texture. Infected fruits remained small and hard and had an off-

flavor.

He et al. (2010) characterized a new variant of ADFVd that caused yellow

dimple fruit formation in ‘Fuji’ apple trees. The viriod was 303nt long and had

84.9% overall nucleotide sequence homology to ADFVd reported from Italy. This

viroid differed from the Italian variant by 47 mutations (38 substitutions, six


55

deletions and three insertions), and most of these mutations occurred on either side of

the central conserved region. A simple and sensitive dot blot hybridization assay

using a digoxigenin-labeled cRNA polyprobe was developed for the simultaneous

detection of six viroids that infect pome and stone fruit trees. The polyprobe was

constructed by cloning sequentially partial sequences of each viroid into a single

vector, with run-off transcription driven by the T7 promoter. All six viroids were

detectable within a dilution range of 5−3

to 5−4

in total nucleic acid extracts from

infected trees (Lin et al., 2010).

Natural occurrence of dapple apple viroid in one of the most popular cultivar

(Starking Delicious) was first recorded in 1995 with an incidence of less than 1

percent in a particular pocket in Shimla district (Thakur et al., 1995, 1996). Later on,

its incidence has been found to increase upto 8-10 percent in some orchards

indicating a serious concern to the growers as dappled fruits become unmarketable

and causes a huge economic loss to the growers (Behl et al., 1998). Handa et al.

(1998) reported the presence of dapple apple viroid in other cultivars of apple in

Shimla district of HP.

2.8.3 Apple fruit crinkle viroid

In 1976, an abnormal graft-transmissible fruit disorder of apple cultivar 'Mutsu’

(‘crispin’) was found in Japan. The symptoms on mature fruits of affected trees were

somewhat similar to dapple apple caused by ASSVd with the addition of fruit

crinkling and blisters on bark. A viroid-like RNA associated with apple fruit crinkle

had a molecular size greater than that of ASSVd and did not hybridize with ASSVd-

cDNA. A difference in molecular size and symptoms from previously described

graft-transmissible diseases of apple led to its classification as a new disease viz.

“apple fruit crinkle (AFC)" (Ito et al., 1993). Ito and Yoshida (1998) ascertained

Apple fruit crinkle viroid (AFCVd) as the causal agent of apple fruit crinkle (AFC)

disease by back-inoculating electrophoretically purified apple fruit crinkle associated

viroid to viroid-free apple trees of cvs. Ohrin, Jonathan, Starking Delicious and

NY58-22 crab apple with chip buds of apple seedlings which had been inoculated by

razor slashing. The nucleotide sequence AFCVd was determined and Koganezawa

(2003) revealed that the viroid belongs to the ASSVd group. It also causes blistering

of bark in delicious cultivars. A study by Koganezawa and Ito (2003) provided

details of the economic impact, symptomatology, host range, transmission,


56

geographical distribution, epidemiology, detection and control of apple fruit crinkle

viroid. Sano et al. (2008) analyzed the genetic diversity of AFCVd, nine apple and

six hop isolates from several locations in Japan. In total, 76 independent cDNA

clones were used for sequencing and phylogenetic analyses. Two major population

clusters were identified. A polymorphism was found in the nucleotide insertion

between positions 142/143 of the AFCVd genome. It was concluded that a genetic

bottleneck caused by vegetative propagation played an important role in the shaping

of viroid populations in a cultivated crop.

2.9 SUSPECTED VIRAL- AND/OR VIROID-LIKE DISEASES

The suspected viral and viroid-like diseases of apple like false sting, green crinkle,

rough skin, star crack are suspected to be caused by agents that are graft

transmissible however, not yet characterized. A brief overview of these is as follows

-

2.9.1 Dead Spur Disease

Dead spur of apple also referred as pink phloem (Klaren and Ketchie, 1979b), spur

death and blind wood is a disease of unknown etiology. The disorder was originally

associated with water stress to the weak-dying spurs which was believed to be caused

by a hormone imbalance. Dead spur infection resulted in the death of the fruiting

spurs on the 3-year and older wood Parish et al. (1983). The primary symptom was

death of the fruiting spurs, usually in the tree's center. Weak spurs were reported to

have smaller and fewer leaves and delayed leaf abscission in the fall. These spurs

could die after only a few leaves form. The phloem of affected spurs may resemble a

pink halo in fresh cross sections (Klaren and Ketchie, 1979a). It was shown to be soil

transmitted and till date considered a low risk malady (Fridlund, 1989). It was

originally associated with Red Delicious cultivars (Ketchie et al., 1978), which

resulted in the death of the fruiting spurs on the 3-year and older wood.

Parish et al. (1983) showed that aminoethoxy vinyl glycine (AVG) applied in the fall

resulted in lesser spur death, and many of the weaker spurs appeared normal. AVG

inhibits ethylene formation and may reduce IAA levels. The results also support

that a growth regulator imbalance may be associated with dead spur. They reported

finding virus-like particles in the proto-xylem of dead spur affected tissue. One

particle is 22 nm wide and 257 nm long. The other particle is 2.8 – 5.5 nm wide and


57

85 nm long. Neither of these particles are claimed to be the incitant of dead spur.

These particles were however, associated found in material from dead-spur affected

trees and not in material from unaffected check trees. Parish et al. (1994) further

reported that an autumn application of an endogenous ethylene inhibitor,

aminoethoxyvinylglycine (AVG), as a foliar spray or as an injection into the trunk

caused remission of dead spur symptoms. Most fruit spurs on treated trees regained

productiveness compared to controls. Repeat application helped to maintain spur

viability.

2.9.2 Apple Blister Bark Disease

Blistering of apple bark has been observed at one or other time in apple orchards.

Two types of blistering symptoms have been reported and referred to as: Apple

blister bark 1 and Apple blister bark 2. In first case initial symptoms appear on bark

as dry, paper thin, orange areas, which slough off. The exposed underlying tissue is

prone to drying, cracking, and peeling. While in case of Apple blister bark 2,

symptoms are identical except for the formation of small cracks in the bark at the

base of spurs and small twigs. Affected areas have greater susceptibility to winter

injury. Internal bark necrosis and twig dieback occurs on limbs that are 1-year old or

older. Leaves and fruits develop no diagnostic symptoms in most apple cultivars. The

symptoms are graft transmissible and till date no major risk due to this has been

reported. However, the causal agent of these symptoms is still unknown (Fridlund,

1989).

2.9.3 Apple Chat Fruit Disease

Chat fruit disease is caused by an unknown pathogenic agent. Posnette and Cropley

(1965) observed that fruit weights were more reliable than visual observations for

diagnosis of chat-fruit disease, especially in young trees. Natural spread of the

disease was reported to be very slow and no vector is known. Infection was not fully

systemic in apple trees. Both healthy and infected trees were propagated from

affected trees, some buds, when used for grafting, transmitted the virus like agent to

the test trees while others did not. Two previously unrecorded effects of chat-fruit

disease, circular spots on the fruit and delayed fruit drop were also observed. This

disease characterized by small sized green fruits is known only in apple and is not

transmitted to other species (Adams, 1988). Electron microscopy of ultra-thin


58

sections of the phloem of apple trees with chat fruit disease revealed mycoplasma-

like organisms (Beakbane et al., 1971).

2.9.4 Apple Bumpy Fruit Disease (India)

Bumpy fruit of apple is attributed to unidentified graft transmissible agent and was

characterized as 'bumpy fruit' (BF) on account of the bumps observed on the fruits.

Bhel et al. (1998) suspected it to be caused by ASSVd variant. It was reported to be a

serious disease in cv. "Golden Delicious" trees in Shimla (HP). Perceptible

symptoms of BF were confined only to the fruits while the trees usually lacked

vigour, bore scarce foliage and had few fruits on each branch. Initially, the fruit

surface developed depressions, at times including a 'false-sting', which became

prominent with bumps upon fruit maturity even though the fruit neither gained much

size nor changed in color as compared to the fruit on healthy trees. Infected trees also

had some fruits which, at maturity, showed hard wart-like swellings with or without

wedges or cracking of varying patterns. The fruits grew slowly in the latter part of

the season. Apparently the fruit quality (sweetness and storage life) was also

affected. The disease incidence was low (0.1 - 1.6%) as most of the orchardists cut

off the affected branches. Yet some growers suggested it to be on the increase.

However, Németh (1986b) reports apple green crinkle agent to be its reason.

Small yellow spots on leaf veins and adjacent tissue resulting in deformation of

blades and fruits becoming malformed with depressions/bumps was also observed.

Transmission was reported to be due to mechanical injury and grafting. A common

symptom observed for both 'bumpy fruit' and dapple apple diseases is the initial

appearance of dark green spots on young fruits. The differences between symptoms

on different cultivars indicated towards the occurrence of strains of the pathogen.

The disease made the fruit unmarketable.

2.9.5 Apple Green Crinkle Disease

Apples infected with green crinkle (AGC), a graft-transmissible disease (Németh,

1986b) was presumed but unproven to be incited by a virus. The apple green crinkle

disease is of worldwide importance reported from North and South America,

Australia, Asia (Israel, turkey, Japan), New Zealand and Europe (Šutić et al., 1999c).


59

Wood (1979) reported it to be especially important in New Zealand as it adversely

affected the major cultivar Granny Smith. Ogawa (1991b) also stated that Granny

Smith, Golden delicious, Graven stein, Mc Intosh and Northern Spy are AGC

susceptible cultivars. However, the disease was not commonly recorded on other

apple cultivars (Thomsen, 1989).

Johnstone and Martin (1968) analysed harvested fruits from Granny Smith

trees, held in cool store for five months. Fruits from infected trees were smaller, had

a higher dry matter content, a greater proportion of protein nitrogen to total nitrogen,

and fewer cells than those picked from healthy trees. Within infected trees the total

nitrogen content decreased as the severity of fruit crinkling increased. Also they

reported a greater incidence of superficial scald in fruit from AGC infected trees.

Fruit symptoms were distinguished from insect stings by the appearance of

discolored/green vascular tissue extending from beneath the pits to the vascular

bundles of the fruit. These strands were not associated with insect stings. Symptoms

intensity varied with the season but was most intense when there were prolonged

cool temperatures after bloom (Johnstone and Sampson, 1986; Limoine, 1987). All

trees exhibiting this serious disease were also reported to be infected with ASGV,

ASPV and ACLSV. It is not known if the disease is elicited by a combination of one

or more of these viruses, a particular isolate of one of these viruses, or a separate,

unidentified pathogen. The disease spread in the orchard appeared to be very slow

and possibly by root grafting. On sensitive cultivars, the fruits developed deep

depressions and distortions that increased in severity as the fruit matured. Sometimes

cracks developed in the pits and crevices. Severe fruit symptoms also appeared on

one or two limbs of an infected tree. There was no leaf symptoms associated with

this disease.

Disease symptom appeared to be enhanced when infected trees were

propagated on precocious rather than seedling rootstocks, especially when they bore

heavy fruit loads. Granny smith and Golden delicious cultivar were reported to be

useful indicator hosts.

2.9.6 Apple False Sting Disease

This disease was often considered a form of apple green crinkle disorder. Fridlund,

(1989) recorded development of a few small depressions on fruits that resembled

insect punctures in mid-season. These depressions were observed to become more


60

pronounced as fruits approached maturity. Unlike insect injury, false sting

depressions exhibited no necrosis of internal tissue. Leaf or bark symptoms were not

apparent.

2.9.7 Apple Flat Limb Disease

Apple flat limb also referred to as apple crinkle wood or apple twisted limb/ twist is a

disease caused by an unknown graft-transmissible agent (Kristensen, 1956). It was

reported from Nova Scotia in 1887 (Hockey, 1943) and later in 1906 from USA by

Clinton (Ogawa, 1991c). It was assumed to be caused by mycoplasma like organisms

by Schmidt (Ogawa, 1991c) while Thomas (1942) considered it to be of viral origin.

Atkinson (1971) reported it as a distinct disease. However, Waterworth and Fridlund

(1989) considered it to have the same causal agent as rubbery wood. They reported it

on all trees of 'Gravenstein' and its red skinned sports. It was latent in a number of

other cultivars. Repeated attempts to confirm this association have failed till now. On

many commercial cultivars, there were no acute symptoms (Ogawa, 1991c), although

fruit quality and yield were adversely affected.

Waterworth and Fridlund (1989) reported no noticeable symptoms on leaves and

fruit. The characteristic symptoms observed on sensitive cultivars were linear

depressions or flattening of shoots or branches, branches fail to develop properly and

remain very pliable and droop under their own weight. On cultivars such as

'Gravenstein' symptoms of flattening of shoots usually appeared on older branches

but was seen in 2- to 3-year-old trees. These depressions become deeper as the tree

matured, causing deep furrows and distortion of older branches. In advance stages,

the bark splits open around the delineated depressions, exposing the wood to

xylophagous fungi. Spindle-like swellings of Gravenstein may be symptomatic of the

disease as reported by Fridlund in 1989 (Figure III: 4). Affected limbs were less

vigorous, easily broken and more susceptible to canker and frost damage. Golden

Delicious cultivar exhibited reduced production (Wood and Cassidy, 1978; Van

Oosten, 1983), and the diseased limbs were very prone to breaking.

2.9.8 Apple Freckle Scurf Disease

Apple freckle scurf was summarized by Németh (1986b) as disease caused by

unknown agent. The bark of diseased trees show small, elevated freckles appear,


61

under which thin necrotic tissue layers are formed. These small freckles crack open,

allowing the bark to scab and peel. Leaves and fruits develop no diagnostic

symptoms.

2.9.9 Apple Fruit Blotch Disease ('Stayman')

It was also described by Fridlund (1989) as an apple disease caused by unidentified

agent showing various symptoms on leaves, fruit and bark. The leaf veins and

adjacent tissue showed cream-colored spotting, while flower petals were deformed

with flecks or rings of red or brown pigment. Fruits also exhibited large red or brown

spots and occasionally thin russet rings 3 to 4 cm in diameter. Blistering of bark on

the trunk and basal region of branches was also recorded.

2.9.10 Apple Necrosis Disease

Apple necrosis disease was thought to be of viral origin viz. Apple necrosis virus. It

was first isolated by Y. Doi (Hull et al., 1989). It has been classified as an Ilarvirus

however, the taxon is not yet listed in the ICTV Report. The genome was reported to

be segmented; tripartite, positive-sense, single-stranded RNA. Minor species of non-

genomic nucleic acid were also found in virions. The multipartite genome was

divided among more than one type of particle and the segments are distributed

between 3 different types of particles. The largest particles contained one molecule

of RNA-1 (sedimenting component B). The medium sized particles contained one

molecule of RNA-2 (sedimenting component M). The smallest particles contain one

molecule each of RNA-3 and RNA-4 (sedimenting component T) (ICTVdB, 2006).

2.9.11 Apple Rough Skin Disease

The nature of the graft transmissible agent that causes rough skin of apple is

uncertain. Symptoms associated with this disease were reported to be influenced by

weather and observed from India, North America, several European countries. The

best expression developed during wet, cloudy spring conditions. Rough, dark brown

corky areas on the fruit result in a scabby appearance. Corky patches may appear as

spots (roundish, elongated and striped) or partial rings. Under cooler growing

conditions star-shaped cracks were formed on the affected areas. Leaf symptoms

consisting of puckering and flecking of the first spur leaf have also been recorded

(Hamdorf, 1989).


62

2.9.12 Apple Rubbery Wood Disease

Apple rubbery wood (ARW), a graft transmissible disease of unknown etiology, was

first reported from England in 1944 (Wallace et al., 1944). The disease was not

transmitted by mechanical inoculation of sap extracts nor by any known insect

vectors. The only means of spread of this disease was by grafting of infected buds

and by propagation of infected rootstocks (Waterworth and Fridlund, 1989). The

causal agent of ARW was initially thought to be a virus (Beakbane and Thompson,

1945), but attempts to isolate virons have failed. Beakbane et al. (1971), using

electron microscopy (EM) later reported the association of phytoplasmas with ARW.

However subsequent studies reported that phytoplasmas could not be detected by

DAPI (4'-6'-diamidino-2-phenylindole) staining in ARW-affected samples (Davies et

al., 1985). In contrast, Minoiu and Craciun (1982) reported EM showed a xylem-

limited bacterium associated with ARW. They reported that the bacterium was

morphologically similar to Actinomycete-like bacteria and showed some similarities

to Rickettsia-like bacteria. Researchers were not able to confirm this observation.

In 1992, Souza and Parish, working with ARW-affected plants from Brazil,

reported the presence of DAPI positive particles, which they claimed to be

phytoplasmas, in the phloem of infected trees. They also reported a remission of

hairy root growth after application of oxytetracycline, and suggested that a

phytoplasma was associated with ARW from Brazil. Another report, from southern

Brazil (Leite and Bleicher, 1993), also claimed that ARW disease was caused by

phytoplasmas, but they provided no experimental evidence to support this claim.

Later studies performed in Germany, also using DAPI staining, with the same apple

material analyzed by Souza and Parish (1992) provided no evidence for the presence

of phytoplasmas (Pollini et al., 1995). Pollini et al. (1995) also reported that no

phytoplasma could be found in ARW-infected tissue using DAPI staining. They also

reported the absence of Rickettsia-like bacteria to be associated with ARW plants

after EM analysis. However, they suggested that it could be due to uneven

distribution of Rickettsia-like bacteria in ARW-infected trees.

More recent studies using phytoplasma-specific PCR assays indicated the

presence of an aster-yellows-type phytoplasma in rubbery wood trees from Italy

(Bertaccini et al., 1998). Neither phytoplasmas nor rickettsia-like organisms (RLO)

were detected in experimentally inoculated Lord Lamborne trees by PCR, using

primers derived from the 16S rRNA gene (Smart et al., 1996), DAPI fluorescent tests


63

or electron microscopy studies in experimentally inoculated Lord Lambourne apple

trees. The results obtained indicated that the apple rubbery wood disease was not

associated with phytoplasmas or RLOs (Pollini et al., 1995).

From India Dhingra and Ahlawat (1973) showed Lord Lambourne and

Victory cultivars to be more sensitive than the others for apple rubbery wood disease.

The virus-like agent was transmitted by grafting to pear, producing symptoms of

chlorotic yellow spots and necrosis on the leaves without causing any rubbery

condition. The symptoms were most noticeable in two-year-old branches, the

characteristic symptom being the abnormal flexibility of stems and branches. One to

three years after young trees are infected the tissues became so soft as to yield to

finger pressure. Diseased trees assumed a drooping habit and could bend as if they

were of rubber; internodes are shortened, annual growth is reduced, sometimes even

stunted. Rootstock productivity, tree vigor and yield may be reduced. There were no

diagnostic symptoms in the fruits or leaves of diseased trees. After 3 to 5 years trees

would again produce normal wood and in some instances the tree recovered and

showed no symptoms. On Golden delicious cultivar ApMV and RW is reported to

cause yield loss of 18-67% (while RW alone can cause upto 46% (Wood and

Cassidy, 1978) loss in yield of this cultivar.

2.9.13 Apple Ring Russeting Disease (Delicious)

Apple ring russet disease (Németh, 1986c) is characterized by mild, light green vein

flecking on scattered leaves during some seasons and fruits showing networks of

russet ring during most seasons. Rings and oak-leaf patterns may also develop on

leaves of some Delicious types. Two types of symptoms were reported to occur on

Golden Delicious cultivar. Type A: produces foliage that exhibited puckering and

flecking, often in rings and lines. Fruit develops slightly sunken rings, white or green

in color, which became yellowish brown (russet) as the fruit ripens. Type B:

develops no leaf symptoms but fruit shows russet areas usually in rings or

incomplete circles. The relationship to leaf pucker and fruit russet symptoms in

Ballarat and fruit russet, leaf pucker, and bark blister in Granny Smith is unclear.

2.9.14 Apple Star Crack Disease

Apple star crack is a disease of unknown origin. It was however, presumed to be of

virus origin (Campbell and Hughes, 1975). Symptoms initiated with the development


64

of bark necrosis around the buds of one-year-old shoots. Bark symptoms varied from

rough bark spots to open cankers. Shoot tips often showed dieback, which was

followed by additional growth from the lower buds. Premature or delayed bud break

and abnormal flowering was observed. During autumn, leaves often became

chlorotic and cupped at the shoot tips. Fruits were reported to be severely

malformed; with characteristic star-shaped cracked spots (scabs) in the skin; small

sized and in some cases, slight depressions and rough skin were also recorded

(Németh, 1986c).

2.10 PHYTOPLASMA DISEASES OF APPLE

Phytoplasmas are cell wall-less and phloem-restricted plant pathogenic bacteria.

They are associated with diseases in several hundred plant species and have been

shown to be transmitted in a propagative manner by sap sucking insect vectors

(Aldaghi et al., 2005). Phytoplasma, formerly known as 'Mycoplasma-like

organisms' or MLOs, are thus specialized bacteria that are obligate parasites of plant

phloem tissue and of some insects. Phytoplasmas are wall-less bacteria of the class

Mollicutes that cause diseases in more than a thousand plant species.

MLOs/phytoplasmas cannot be cultured in vitro in cell-free media. They are

characterized by their lack of a cell wall, a pleiomorphic or filamentous shape,

normally with a diameter less than 1 micrometer, and their very small genomes.

Phytoplasmas require a vector to be transmitted from plant to plant, and this

normally takes the form of sap sucking insects such as leaf hoppers in which they are

also able to replicate. They were first discovered by Doi et al. (1967).

Molecular diagnostic techniques for the detection of phytoplasma began to

emerge in the 1980s and included ELISA based methods. In the early 1990s, PCR-

based methods were developed that were far more sensitive than those that used

ELISA and RFLP analysis allowing the accurate identification of different strains

and species of phytoplasma (Chen et al., 1992). They were classified based on

sequence homologies of the 16s rRNA gene (Seemüller et al., 1998; Gundersen et

al., 1994), but recently rules have been established to define a “Candidatus”

phytoplasma at species level (Seemüller and Schneider, 2004; IRPCM, 2004). More

recently, techniques have been developed that allow for assessment of the level of

infection. Both Quantitative-PCR and bioimaging have been shown to be effective


65

methods of quantifying the titre of phytoplasmas within the plant (Christensen et al.,

2004).

2.10.1 Apple Proliferation Phytoplasma (Phytoplasma AP-MLO)

Apple trees show a number of different disease syndromes many of them are of

uncertain etiology. There has been a good evidence of phytoplasma infection

associated with apple proliferation (AP) disease (Brcˇa´k et al., 1972; Lorenz et al.,

1995; Brzin et al., 2003). Rickettsia-like bodies were observed by electron

microscopy in leaf pedicels of Golden Delicious apple with proliferation disease

(Petzold et al., 1973). AP was first described in Italy in the fifties (Rui, 1950, Refatti

and Ciferri, 1954). The phytoplasma associated with this disease has been recently

classified as ‘Candidatus Phytoplasma mali’ (Seemüller and Schneider, 2004). This

is one of the most important phytoplasma diseases of apple, affecting almost all

cultivars, reducing size (by about 50%), weight (by 63-74%) and quality of fruit, as

well as reducing tree vigour and increasing susceptibility to powdery mildew

(Podosphaera leucotricha) (De Salvador et al., 2007). Apple proliferation (AP) is

one of the most serious phytoplasma diseases of apple trees in Europe and causes

considerable economic losses mainly by decreasing the size and quality of fruits

(Frisinghelli et al., 2000). An interaction between apple rubbery wood disease and

apple proliferation was also reported stating that the former promoted transmission of

the latter. Characteristic AP symptoms such as proliferation or premature

development of auxiliary shoots (witches’ brooms), reduced flowering, phyllody,

enlarged stipules, reduced leaf lamina, leaf rosettes, chlorosis, yellowing and early

leaf reddening were also recorded (Kartte and Seemüller 1988). An increase of

phenolic compounds and hydrogen peroxide in host plants infected by phytoplasmas

has also been recorded (Musetti et al., 2000; 2004; Junqueira et al., 2004).

The economic relevance is due to smaller fruit size, lower fruit quality and

overall yield decreases (Baric et al., 2008). Fruits of apple cv. Jonathan affected by

AP phytoplasma, showed marked reduction in fruit size; longer, thinner peduncles

and shallower calyx end and peduncular cavities. AP was proven to be transmitted

through infected grafting material (Kartte and Seemüller, 1988) and sap-sucking

insect vectors (Frisinghelli et al., 2000; Tedeschi and Alma, 2004; 2006). Baric et al.

(2008) reported that root connections had role for the spread of AP phytoplasma at

least in older orchards and between trees on vigorous rootstocks.


66

2.10.2 Apple Sessile Leaf Phytoplasma

Jomantiene and Davis (2005) described a new disease of apple found in the

Kaisiadorys region of Lithuania. With symptoms of leaf yellowing, shoot

proliferation and a previously undescribed symptom of sessile leaf, where leaves

were directly attached to the trunk. These symptoms differed from those of apple

proliferation (AP), with enlarged stipule, witches' broom and bronze-reddish

discoloration of leaves (Baric and Dalla-Via, 2004). DNA extracted from leaves,

with and without symptoms, was used in PCR with phytoplasma rDNA universal

primers P1/P7, as previously described by Valiūnas (2003). Phytoplasma infection

was confirmed and analysis revealed that this phytoplasma, termed apple sessile leaf

(ApSL) phytoplasma, belonged to 16SrI-B ('Candidatus Phytoplasma asteris')

subgroup. The results of this study clearly distinguish ApSL from AP phytoplasma,

'Ca. Phytoplasma mali', a member of group 16SrX.

2.11 MANAGEMENT

Viral diseases in fruit trees present a potential danger that in the long run would

injure the fruit industry, the planting stock industry (nurseries) and consumers. Apple

is propagated through grafting. Most of the major apple virus (es), viroids and virus

like agents spread due to infected planting material, i.e. they are graft transmissible.

Once a plant is infected, it cannot be cured and serves as source for further spread.

This results in poor health of trees in orchards. Detrimental effects of virus infections

on growth and yield are reported on apple trees. Viruses can cause huge losses to

crop even without producing any visible symptoms (Cembali et al., 2003). Losses

caused by viruses are both qualitative and quantitative. Like fungal and bacterial

pathogens, virus diseases cannot be managed by chemical treatments which can

constrain only insect vectors that may spread viruses. Scouting and treating for virus

transmitting vectors (insects, fungus etc.) would be necessary throughout the year,

costing growers in time, labor and materials for a control strategy that is not

adequately effective. Moreover, viral infection passes to successive generations

through vegetatively propagating planting material in apple and other pomaeceous

and prunus fruits, resulting in decline in plant health and low productivity over a

number of years.

2.11.1 Classical Approach


67

The apple is an ancient crop that has been under a continuous selection since

antiquity. Intensive methods of production, particularly high-density orchards with

some genotypes can be expected to have a profound effect on the future apple

industry of the world. However, certain restraints to conventional plant breeding that

are especially limiting in tree fruits with long juvenile period and that are represented

by unique, highly selected genotypes need to be dealt with. These include the

reliance on naturally occurring variation which may be unavailable, or more likely

found in very primitive and unadapted material, restraints due to the inability of the

sexual system to incorporate genes from nonrelated species and especially the

inability of the sexual system to incorporate small changes without recombination

resulting in the loss of desirable unique combinations; the difficulty of selection in

detecting infrequent or rare recombinations. Also the dependence of conventional

breeding upon time to generate cycles of recombination, space to grow the necessary

population to recover superior recombinants, and resources to be able to select,

identify, and evaluate desirable recombinants. Some of these limitations may be

overcome by novel strategies from advances in biotechnology.

Cultivation of virus resistant genotypes are often restricted by the availability

of a limited germplasm pool for a source of virus resistant as opposed to the wide

host range of viruses in addition breeding programme is laborious and time

consuming, especially for the crops with long generation cycles. The conventional

breeding for resistant cultivars using available germplasm is a long term strategy and

production of virus resistant cultivars may take decades. Haniuda et al. (1984) in a

study of crosses between the rootstock parental material Seishi (Malus prunifolia var.

ringo), susceptible to ACLSV, Sanashi 63 (M. sieboldii), susceptible to ACLSV and

ASPV, and virus- resistant dwarf rootstocks, M9, M26 and M27, that susceptibility

to ACLSV was concluded to be controlled by a single major gene. To reduce the

time genetic engineering approach has been used to generate resistant.

Cross protection using a milder virus strain can also check the major loss by virulent

strains of related and/or unrelated virus (es), however some resources have to be

sacrificed for mild infection also. In 1976, Marenaud et al. analyzed the possibilities

of interactions between four purified strains of ACLSV on two indicators, and the

results were recorded with regard to the appearance of line pattern on Malus

platycarpa and bark split on Prunus domestica P 707. Results greatly depended on

the host plant and the nature of the strains. On M. platycarpa, no cross protection


68

was observed, and line pattern symptoms did develop whatever the strain

combination was. On the contrary, inoculation of P. domestica P 707 with a mild

strain (unable to induce bark split symptoms) always prevented the plant from

developing symptoms when super infected with a strain normally inducing severe

bark split. Thomsen (1975) divided twelve varieties into four categories according to

the effect of ApMV infection on the trunk girth of trees inoculated with a severe

virus strain alone or severe or mild strains together. In the first group, which included

Belle de Boskoop [Boskoop Beauty], Cox's Orange Pippin and James Grieve,

inoculated and control trees grew equally well. In the second- McIntosh and Stark

Earliest, the control trees grew best. In the third, comprising Graasten (Gravenstein)

and Spartan, the mild strain afforded protection against the severe strain, but the

control trees grew best. In the fourth, containing Guldborg, the trees inoculated with

the severe strain grew more poorly than the controls and the cross-protected trees.

Selection, propagation and distribution of true to type disease free and virus-

indexed quality planting material is foremost for establishing healthy and profit

earning orchards. Clean horticulture practice as simple as rinsing the secateurs and

grafting knives with spirit before using can check the spread effectively.

2.11.2 Biotechnological approach

2.11.2.1 Tissue culture/Micropropagation techniques

Information on tissue culture of apples has been reviewed by Zimmerman (1988,

1991). The most effective and practically viable used measure so far for the virus

eradication is use of virus free propagation material obtained by employing the

meristem tip culture alone or in combination with chemo and thermotherapy. Engel

(1990) studied the importance of virus free planting material in integrated fruit

production by observing the behavior of virus free and virus infected apple and pear

trees and found that the virus free trees were more vigorous, had higher fruit yields

and better quality fruits than virus infected trees and they did not required additional

treatments with fertilizers and growth regulators. They also found that virus free trees

were also better for successive replanting.

The technique of virus elimination pioneered by Morel and Martin (1952) was based

on the assumption that virus particles are unevenly distributed in the plants and their

titer decreases as the meristmatic dome of vegetative shoot apex is approached

(Limmaset and Cornuet, 1949). The apical meristem domes were thus considered


69

free from virus particles. Virus free production of plants is necessary from the point

of view to prevent further spreading of virus. Use of virus free propagative material

will prevent further dissemination of viruses in the field and thus can prevent long

term losses.

To restore the health of plants with latent virus infection Shvartsbakh et al.

(1977) treated the plants by keeping the crown at 38 plus or minus 1 deg C and the

roots at 18-20 deg , with illumination at 6,000 lux/m2 (30% from incandescent and

70% from luminescent lamps) for 16 h/day and RH 70-80%. Treatment was

continued for 4-5 weeks and followed by tip propagation. Plants of the cultivars

Wealthy Pepin saffron and the rootstock EM-IX from apple ASPV and ACLSV were

obtained. Inactivation of viruses was maximum with better plant growth in the heat

chamber. They reported that best results were obtained on grafting in the second half

of summer.

Of the viruses infecting Malus spp. ACLSV can be relatively easily

eliminated by short periods of heat treatment (Campell and Best, 1964; Welsh and

Nyland, 1965; Campell, 1968; Cropley, 1968) and by apical meristem cultivation

(Gabova, 1989). Blattny et al. (1975) heat treated young grafts of 19 apple cvs after

bud break were grown in a sawdust-peat (2:1) mixture at 37 deg C for 3 weeks. The

shoot tips of the heat-treated plants were re-grafted onto healthy rootstocks and kept

in moist sawdust in polyethylene bags at 21 deg for 10 days. They were then virus-

tested using R 12740-7A, Spy 227, Virginia Crab or Lord Lambourne as indicators.

Altogether 79% of the shoot tips were free of chlorotic leaf spot virus, 75% of Spy

decline, 36% of Spy decline + stem grooving, 95% of apple mosaic and 49% of

rubbery wood. Larsen (1977) analyzed growth patterns of buds of several virus-free

(by heat therapy) and of one virus-infected clone of each of 10 apple cvs were

grafted onto virus-free rootstocks of MM.106. Similarly, scions of clones of 5 cvs

were grafted on MM.106 in spring under glass. Trees of virus-free clones generally

grew taller and had thicker stems and, with some cultivars produced more and longer

laterals than infected material. However, there was great variation in growth response

between individual trees.

Apple shoot cultures systemically infected with the virus were grown on

Murashige and Skoog medium to which the virus inhibitor ribavirin had been added

were virus-free after the first and second treatment periods. The resulting rooted

shoots remained virus-free during subsequent transfers to ribavirin-free medium, to


70

greenhouse conditions, and to the field. Results indicate that ribavirin treatment of

cultured shoots is a reliable and simple method for eliminating the virus. (Hansen,

1979; Hansen and Lane, 1985).

Desvignes (1980) demonstrated that heat therapy may be applied to separate

virus complexes and treatment of infected apple cultivars permits the isolation of

apple chlorotic leafspot, apple Spy decline or apple stem grooving viruses. He

concluded that since Spy decline, Pyronia decline, apple stem pitting and quince

sooty ringspot cannot thus be separated, they may be caused by the same virus. Cross

protection may be used to compare diverse symptoms.

In a study by Baumann and Louis (1980), heat treated virus-free (VF) M.9

apple rootstock clones were compared with tested (T) M.9 clones free from most of

the important viruses but still containing some latent viruses (e.g. ACLSV, ASPV).

Only in easy-rooting clones did VF plants root slightly better than T plants. VF

plants grew a little better and their losses during the winter and to Cytospora sp. were

less than with T clones. Leaves of VF clones were larger and darker green than those

of T clones but there was no difference in shoot and internode length. VF clones had

a higher Ca-uptake and required less N than T clones to produce similar growth.

Legrand and Verhoyen (1983) demonstrated cultural improvement of virus free P.

blireana, P. cerasifera cultivars Trailblazer and Woodii, Helen Borchers peach and

P. triloba, naturally infected by a ACLSV and PNRSV complex. The material was

cured by thermotherapy before grafting onto Brompton plum rootstock. In

comparison with infected plants similarly grafted, graft recovery rates were on

average 112% higher. Symptoms disappeared and growth was improved, average

increases in total height from the graft union being 33%; girth at the union 17%; total

weight of the trees 84%; and mean length of the year's twigs 20%.

Gabova (1987) obtained the apple cultivars, Molis Delicious and Belle

Golden, free from ACLSV and ASGV by in vitro meristem grafting. Thermotherapy

and chemotherapy were carried out on in vitro-propagated sweet cherry (Prunus

avium) and P. mahaleb shoots infected by PNRSV, Prune dwarf ilarvirus and

ACLSV by Deogratias et al. (1989a). They reported chemotherapy as all the more

efficient as the plants underwent intensive vegetative multiplication, however they

also concluded that success depends on the kind of virus and on the type of genetic

plant material. By using a medium rich in hormones, particularly in cytokinins, an

intensive vegetative multiplication was obtained. This active proliferation produced a


71

notable decrease in the virus content of shoots obtained by subculturing. It seems that

in vitro culture of these infected clones took advantage of the competition between

cellular and viral multiplication. The hormonal composition of the culture medium

seems to play a leading part in this competition (Deogratias et al. 1989b).

Arai et al. (1990) observed that if either a size-controlling rootstock or a

cultivar scion carrying infection by ACLSV, the Malus prunifolia var. ringo

rootstock developed necrosis and pitting of the inner bark. Seedlings of M.

scheidekeri, susceptible to the virus, were planted in an orchard with soil infested

with a high density of Rosellinia necatrix and Helicobasidium mompa and then half

were inoculated with ACLSV. Trees with the virus were more susceptible to the 2

root pathogens than were virus-free trees. In another orchard where H. mompa was

predominant, trees with virus-induced necrosis on the M. prunifolia var. ringo stock

were more severely affected by the fungus than were virus-free trees.

Engel in 1990 gave the importance of virus-free plant material in integrated

fruit production trees and found that virus free trees produced by tissue culture were

more vigorous, had higher fruit yields and better quality fruit than virus-infected

trees, and did not require additional treatments with N-fertilizers and growth

regulators. Virus-free trees were also better for successive replanting, and could be

planted at lower densities than virus-infected trees. An in vitro virus elimination

programme for Malus and Prunus spp. was carried out using in vitro thermotherapy

and meristem tip culture. After the treatments, Malus sp. was tested for ApMV,

ASGV and ACLSV, and Prunus for PPV, PNRV, PDV, ApMV and ACLSV by

ELISA and immuno tissue-printing. PPV, PNRSV, ASGV and ACLSV were

detectable after 4 years of in vitro culture. PDV was found in an 8-month old Prunus

culture. ApMV was lost after 2.5 years of in vitro culture. PPV could be detected in

leaves after 20d of thermotherapy, but not after meristem culture. ASGV was either

positive after 33d of thermotherapy or reduced below the threshold level for reliable

serological detection. ASGV titre significantly increased 6 months after

thermotherapy and meristem dissection. No ACLSV positives were found after 33d

of thermotherapy, and shoots grown from meristems of main shoots were free of

ACLSV 6 months after, whereas shoots from axillary meristems were infected

(Knapp et al., 1995b).

Cieślińska and Zawadzka (1999) used thermotherapy and chemotherapy

using Virazole (ribavirin) in various concentrations were applied to eliminate


72

ACLSV and ASGV from apple shoots, ACLSV from pear shoots. The majority of

the plantlets of these species treated with high temperature survived. Thermotherapy

allowed elimination of ACLSV from 66.7% of apple shoots and 24% of pear shoots.

Virazole in concentrations 25-50 mg/l eliminated ACLSV from the majority of

treated apple and pear shoots. 50 and 100 mg/litre ribavirin reduced the growth of

shoots and caused chlorosis of plantlets and apex necrosis. Ribavirin at a

concentration of 100 mg/l was phytotoxic for 65-86% treated shoots, depending on

plant species. Thermotherapy combined with 100 mg/l ribavirin caused the death of

the majority of apple shoots and all pear shoots.

Cieślińska (2002) subjected micropropagated shoots of pear cv. Pierre

Corneille plants infected with ACLSV to heat therapy at 36 degrees C or/and

chemotherapy using ribavirin (Virazole; at 10, 25, 50 or 100 mg/litre) to eliminate

the virus. The presence of virus in recovered plants was evaluated by ELISA

technique during one year after therapy. The majority of treated pear shoots survived

thermotherapy. The effect of chemotherapy depended on the concentration of

Virazole. Shoots treated with the 50 and 100 mg Virazole/litre showed chlorosis and

apex necrosis. The highest concentration of this compound appeared to be highly

phytotoxic to 77% of pear shoots. Almost all shoots of pear treated with high

temperature and 100mg Virazole/litre died. Thermotherapy of Pierre Corneille shoots

enabled to eradicate ACLSV from 22% of plantlets. ELISA did not show any

presence of ACLSV in 78 and 88% of pear shoots treated with Virazole in

concentrations of 25 and 50 mg/l, respectively. The combination of thermotherapy

and chemotherapy gave no better results than chemotherapy alone. Lukicheva and

Mitrofanov (2002) successfully eliminated PNRSV and ACLSV from cherries

(Cerasus vulgaris [Prunus cerasus]) after 20 and 40d of thermotherapy, respectively.

For plums (P. domestica), PNRSV was successfully eliminated after 30 days of

treatment. Conditioning was done at 37+or-1 degrees C, under a 16-hour

photoperiod. The temperature was decreased by 10 degrees C during night hours.

O' Herlihy et al. (2003) studied the titre of ACLSV and PNRSV. For both

viruses titer in the roots of autotrophically-grown plants was significantly higher than

in haulm tissue from heterotrophic cultures. Ribavirin incorporation resulted in

elimination of both viruses. The efficacy of ribavirin in elimination of ACLSV was

increased by increasing the concentration of cytokinin in the medium in parallel with

decreasing the concentration of ribavirin. Qu et al. (2003) standardized an improved


73

method for virus elimination. A total of 16 apple cultivars with ACLSV, ASPV and

ASGV latent viruses were used. Some plants were potted, whereas others were

planted under plastic in a greenhouse. At the 4-5 leaf stage, the plants were heat

treated; potted plants were treated in a glass cabinet and the others were treated in a

shed. Heat treatment for 26 days at 37 degrees C in the shed achieved elimination

rates up to 72.3%, while many of the treated potted plants died. Heat treating tube

plantlets in an illumination cabinet for 30 days at 38 degrees C provided 100% virus

elimination with a survival rate of 13.3-60%.

Yuan et al. (2005) showed that in cherry only the 0.3 mm stem-tips cultured

could eliminate ACLSV and PNRSV. In a study by Wang et al. (2006), in vitro

thermotherapy was carried out for ASGV and ACLSV the two major viruses of pear.

Treatement was given at 37 degrees C for 25, 30 and 35 days followed by

subculturing of meristem stem tips of different sizes were used to eliminate ASGV

and ACLSV from pear plants. Virus titers in heat-treated shoot tips were evaluated

by ELISA testing of regenerated plants. Results showed that thermotherapy for 35

days significantly decreased the titer of ASGV and ACLSV in cultures regenerated

from tips of main and axillary shoots, especially in those from explants 1 mm in

length from the tip of meristems. Dot-blot hybridization of biotinylated cDNA

probes derived from ACLSV and ASGV was used to confirm these viruses in crude

tissue extracts of in vitro-grown pear plants. Results of PAS-ELISA and dot-blot

hybridization showed that high virus elimination efficiency was achieved by a

combination of thermotherapy for 35 days and in vitro culture of 1 mm meristem

tips. It was also concluded that dot-blot hybridization could detect viruses with a titer

below the threshold level of ELISA. These results indicate that dot-blot hybridization

is a useful tool for large-scale surveys of viruses, which facilitates the production of

virus-free propagation materials in certification and sanitation programs.

Sedlak et al. (2007) and Paprestein et al. (2008) used apple cultivars 'Idared'

and 'Sampion' and pear cultivars 'Alexander Lucas' and 'Max Red Bartlett' for

application of in vitro thermotherapy. Selected cultivars were successfully multiplied

as in vitro cultures. Multiplication coefficient of four used genotypes ranged between

2.3 and 2.9. After the end of thermotherapy, one cultivar of pear 'Alexander Lucas'

(variant 22) and one cultivar of apple 'Idared' (variant 6) was free of all tested viruses

after RT-PCR testing and transfer to ex vitro conditions.


74

From India, Bhardwaj et al. (1998) used apple meristems ranging in size from

0.1 to 1.0 mm were cultured on MS medium supplemented with benzyladenine (1

mg/l), IBA (0.1 mg/l) and GA3 (0.1 mg/l) for virus free production. As the meristem

size increased, the rate of meristem survival and shoot regeneration increased.

ApMV could be detected in cultures raised from meristems of >0.2 mm. Complete

virus inhibition was observed with hot water treatment of wood scions at 47 degrees

C for 30 minutes and 50 degrees for 15 minutes. Hot air treatment of potted plants at

37 degrees for 4 weeks and 40 degrees for 2 weeks also resulted in complete virus

elimination.

Wang et al. (2010) investigated the distribution patterns of ASGV and

ACLSV in in-vitro cultured pear plants using in situ tissue-printing hybridization

(TPH) and tissue blotting immunoassay (TBIA) to detect viral RNAs and coating

proteins. Both ASGV and ACLSV showed high concentrations in the tip of the pear

shoots and lower concentrations in the middle stem. The highest viral RNA titers

were found in the phloem parenchyma of vascular bundles. The heat treatment was

greatly effective to reduce virus titers in the shoot tips. No viral RNAs of ACLSV

and ASGV were detected in less than 2mm and 0.5 mm long tips, respectively.

In a study from 1998-2008 by James (2010) it was confirmed that the

antiviral chemical combination of ribavirin and quercetin (10 μg/mL of each) was

effective for the elimination of ASGV from apple. ASGV was also reported to be

eliminated from infected N. occidentalis cultures by this treatment.

2.11.2.2 Transgenic approach/ Genetic engineering

Recombinant DNA has been especially attractive to fruit breeders because it offers a

way to overcome the limitations of the sexual system by permitting the introduction

of foreign genes and more important makes it possible to introduce small discrete

defined changes into established genotypes. The use of molecular markers will

facilitate selection and provide the ability to identify clones with a great deal of

precision for cultivar protection and perhaps to tag clones with a genetic brand.

Recent advances by a new generation of fruit breeders have demonstrated that these

new technologies are feasible in apple. However, it remains clear that these new

technologies will not replace but will merely complement conventional breeding.

Most of the advances in DNA transformation will in the end involve improvements


75

due to the insertions of single genes, which represent a conservative approach to

improvement.

2.11.2.2.1 CP Mediated Resistance

Among the pome and stone fruits coat protein (CP) mediated resistance has been

obtained

against Plum pox virus. Transgenic plants were obtained by co-cultivation with

Agrobacterium tumefaciens strain containing various binary plasmids and coat

protein gene of Plum pox virus (Machado et al., 1993; Scorza et al., 1994; Lo´pez-

Moya et al., 2000; Revalonandro et al., 1997) further studies and on-field trials are

being conducted (Revalonandro et al., 2000).

2.11.2.2.2 RNAi for virus resistant plants

In addition to the classical methods for producing resistant germplasm and cultivars

genetic engineering has turned out to be a promising approach. Genetic

transformation offers a means of incorporating new virus resistance traits into

existing desirable plant cultivars. RNAi technology at present is an effective

management strategy to control plant viruses.

There was no convenient system for the expression of foreign genes in fruit

trees like apples. Li et al. (2004) could express the GFP gene in leaves of apple

seedlings inoculated with ALSV-RNA2 vector containing a GFP gene. It is believed

that the ALSV vector would be available for the expression of foreign genes in apple

trees and serve as a virus vector. Pathogen derived resistance uses genes from

pathogen itself to create transgenic which on infection with that particular pathogen

and/or some related /unrelated pathogens confers resistance to the plant. In case of

ACLSV, P50 (i.e. ACLSV-MP) has been identified as suppressor protein and is

reported to cause systemic silencing in Nicotiana benthamiana line 16c, indicating

that functions as a suppressor of RNA silencing (Yaegashi et al., 2007b). MP-derived

resistance using defective ACLSV-MP has also been reported to interfere

specifically with the functions of the MP encoded by GINV and vice-versa (Isogai et

al., 2003; Yoshikawa et al., 2000, 2006b). GINV is another member of Trichovirus

genus however, the resistance was not due to gene silencing and was attributed to

loss of activity of the movement proteins (MP) due to complex formation.


76

Experiments have proved RNAi technology to be suitable to control infection by a

variety of plant viruses. PPV is the causal agent of sharka or plum pox, the most

serious disease on woody trees of the genus Prunus (Tenllado et al., 2004). Similarly

the vole-resistant apple rootstock ‘Novole’, which is sensitive to ASGV, transmits

this sensitivity to more than half its seedlings. The incorporation of virus resistance

into plants is one of the successes of genetic engineering (Mauren et al. 1992) and

this approach is under investigation in apple (Grumet, 1994).

Because of their highly ordered structure, mature viroid RNA molecules were

assumed to be resistant to degradation by RNA interference (RNAi). However,

recently Schwind et al. (2009) developed transgenic tomato plants expressing a

hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd)

sequences exhibit resistance to PSTVd infection. Resistance was correlated with

high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the

plant. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21-

and 24-nucleotide siRNAs of both (+)- and (-)-strand polarities along the PSTVd

genome. This suggests that RNAi could also be employed to engineer plants for

viroid resistance, as has been well established for viruses.

Thus losses caused by viruses and virus like pathogens are both qualitative and

quantitative. Unlike fungus and bacteria it is difficult to manage viruses by chemical

control and conventional methods used for other pathogens.

2.12 Relevance of work and Future prospect:

Fruit tree viruses were often regarded as minor causal agents for tree diseases since

viruses infecting fruit trees frequently cause few symptoms (Kim, 1999). From the

review, it is evident that in the Indian scenario also viruses in horticultural and

perennial crops like apple, other pome and stone fruits have not been considered

important. The virus-infected cells are generally not killed but they allow viral

replication and production of progeny viruses that further infect tree. Unlike virus-

infected crops and fodder that still can be used, fruit trees and ornamental crops can

be totally lost or can cost a massive economic damage by adversely affecting fruit

quality and/or productivity. However, large scale production and major economic

returns from apple in other temperate fruit growing areas of the world led to indepth

analysis, characterization and research on ACLSV and other apple

viruses/viroid/phytoplasmas/graft transmissible agents. In India, mainly the lack of


77

awareness about the viral/virus like agents, their identification, negligible reports of

losses, latent infections and little knowledge of management practices contribute to

silent spread of these viruses. Summarizing the review it can be stated that initial

work of identification, molecular characterization and development of economical

diagnostics are the steps that would eventually lead the research on ACLSV and

other pome-stone fruit viruses from infancy to maturity. The work would provide

basis for understanding pathogen (virus) gene function analysis, host-pathogen

interaction studies, understanding resistance, development of virus free transgenics

and use of virus as plant vectors. The elementary work would also in turn generate

awareness about the viruses of horticultural crops like apple and trigger research for

ways to manage the evil.

Documents

REVIEW OF LITERATURE - Information and Library …shodhganga.inflibnet.ac.in/bitstream/10603/7047/7/07... · · 2015-12-04Review of Literature 4 ... In fruit crops, viruses are