Results - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/7047/9/09_chapter 4.pdf · Results 122 Fig 4.1 I: (A) Chlorosis and leaf puckering in cv. Red Chief apple seedling in

Results

120

RESULTS

4.1 SURVEYS AND VIRUS INCIDENCE ON POME AND STONE FRUITS IN HP

AND J&K

A total of 40 orchards were surveyed (2007-2009) in 24 different locations for the incidence

of ACLSV mainly in apple along with other pome and stone fruit plantations. From each

orchard 10-15 trees (7 symptomatic, 3 healthy) were selected at random and 8-10 samples of

leaves were taken from each tree. On an average 290 apple, 15 quince, 35 pear, 50 plum, 60

peach/nectarine, 53 apricot (wild and cultivated), 20 almond, 100 cherry (wild and cultivated)

trees were analyzed. Apart from these, some wild relatives such as wild Himalayan cherry,

wild apricot, wild peach (which are used as rootstocks for the cultivated stone fruits) and

plants were also tested for ACLSV. Seedlings from four nurseries of HP were also tested

4.1.1 Based on Visual Symptoms

Virus symptoms vary according to their occurrence on the leaves (chlorotic alterations,

deformations, enations, necrosis), fruits (alteration of shape, color, size, chemical

composition), and wood trunk (pits, different diameter between scion and rootstock, graft

union necrosis). During the surveys most of the virus like symptoms of puckering, curling,

mosaic, oak leaf patterns, shot holes, chlorosis were found on some of the apple, other pome

and stone trees indicating towards the presence of virus infection (Fig 4.1 I, II, III).

Malformations due to reduction in leaf size and chlorotic rings or line patterns on foliage and

asymmetric leaf distortion were observed randomly in most of the apple orchards. Symptoms

of mosaic (mostly in pollinizer cultivars Golden Delicious, Granny Smith and Tydeman’s)

and leaf deformation due to curling were very predominant on apple leaves (Fig 4.1 I, II).

Symptoms of fruit deformations and discoloration in apple were observed mostly from

Shimla areas. The symptoms were present discontinuously (one branch healthy other

symptomatic) in many apple trees. Pits and grooves on bark and trunk area in apple trees were

rarely observed (Fig 4.1 II). However, most of the apple trees were apparently healthy and no

diagnostic symptoms were observed. Among other pome and stone fruits shot holes, mosaic,

chlorosis, puckering and necrosis were more common (Fig 4.1 III). ACLSV infection is

known to be latent in most apple cultivars. An estimated disease incidence of ACLSV

infection on apple, other pome

Results

121

Table 4.1 Different hosts and locations surveyed

*All – apple, apricot, almond, cherry/ wild Himalayan cherry, pear, plum, peach/nectarine,

quince

S. No. Location District State Orchards

surveyed

Samples Collected

1. Palampur Kangra

HP

2

Nurseries

All

2. Kaza Lahul &

Spiti 3 Apple

3. Salooni Chamba

3 Apple, plum, pear, quince

4. Tissa 2 Apple, plum

5. Sangla

Kinnaur

1 Apple, peach

6. Kalpa 1 Apple

7. Kinnaur 3 Apple, apricot, almond

8. Kotkhai

Shimla

2 Apple, cherry

9. Kotgarh 2 Apple, cherry

10. Narkanda 1 Apple

11. Sarahan 1 Apple, peach, plum

12. Annu 1 Apple

13. Nihari 1 Apple

14. Theog

Mandi

2 Apple, plum

15. Dobi 1 Apple, plum

16. Thunag 2 Apple, plum

17. Bajaura

Kullu

2 All

18. Seobagh 1 All

19. Manali 1 All

20. Nauni Solan 2 All

21. Renuka Sirmaur 1 Apple, peach, plum

22. Jammu Jammu

J&K

1 Cherry, peach, pear, plum

23. Pulwama Pulwama 1 Apple, cherry, plum,

almond 24. Gandarbal Gandarbal 1 Apple, cherry, plum

25. CITH Srinagar 1 All

26. Attari Amritsar Punjab 1 Peach, plum

Results

122

Fig 4.1 I: (A) Chlorosis and leaf puckering in cv. Red Chief apple seedling in Palampur

nursery; (B) Chlorosis and leaf deformation on cv. Royal Delicious leaves from Nauni; (C)

Severe mosaic and deformation in Golden Delicious on leaves, (D) oak leaf-like pattern

observed on apple leaf at Srinagar; (E) Inter-vein chlorosis on a apple leaf at Theog; (F)

Severe mosaic, vein clearing and leaf deformation recorded at Bajaura.

Results

123

Fig 4.1 II: (A) Necrotic spots and shot holes on leaves of cv. Golden Delicious from

Seobagh; (B) vein clearing on cv. Tydeman’s at CITH; (C) Severe chlorosis on cv.

Starkrimson compared to a healthy leaf at IHBT, Palampur farm; (D) Chlorotic spots on the

cv. Top Red from CITH; (E, F, G) Pits and grooves seen in the tree trunk at Theog, Salooni

and Gandarbal.

Results

124

Fig 4.1 III: (A) Apple fruit deformation from Shimla, (B) Leaf chlorosis and fruit

deformation in quince at Salooni, (C) Chlorotic spots on peach at Palampur (D) leaf chlorosis

in plum cv. Kala Amritsari at Attari, (E) Pox-like symptoms on apple from Mandi, (F) Leaf

yellowing, necrotic spots and shot holes in plum cv. Santa Rosa at Bajaura.

Results

125

Table 4.2: Incidence of viral infection on the basis of visual symptoms

and stone

fruits based

on visual

symptoms would be misleading. However, a general estimate of virus infection on pome and

stone fruit trees based on visual observation is listed in Table 4.2

4.1.2 Based on Enzyme Linked Immunosorbent Assay (ELISA)

Preliminary detection of the symptomatic plants and randomly selected symptomless/healthy

plants from each orchard were subjected to DAS-ELISA for the diagnosis of ACLSV. Other

important viruses infecting apples viz. ApMV, ASGV, ASPV, PNRSV, PPV were also tested

as ACLSV in nature occurs mostly in mix infection. ACLSV detection was more reliable in

spring season (March-April) using flower petals, buds and young leaves in apple, other pome

and stone fruits. Even ACLSV positive samples gave ambiguous or negative ELISA readings

in dormant season (July- September). ApMV was also best detected with flowers while

ASPV and ASGV could be detected throughout the year using mature leaves and to some

extent using bark also.

4.1.2.1 Apple and other Pome Fruits:

The infection was very widespread even at nursery level as ACLSV was confirmed in about

14 commonly grown apple cultivars out of the 18 tested from two of the Palampur nurseries

(Table 4.3). For each of the cultivars 5 plants were tested and if atleast one sample tested

positive (greater than two times the absorbance value than

Table 4.3: ELISA results of apple cultivars tested for various viruses from different

nurseries (P1= CSK HPKV, Horticulture nursery, Palampur; P2= State Horticulture

Department nursery, Palampur; P3= University of Horticulture and Forestry, Solan; P4=

CSK HPKV Regional Research Station, Bajaura.)

S.No. Cultivars % Disease incidence based on visual

symptoms

1. Apple 50

2. quince 25

3. Pear 40

4. Peach/ Nectarine 60

5. Almond 10

6. Apricot 15

7. Plum 40

8. Cherry 30

Results

126

S.No. Cultivar Place ELISA

ACLSV ASPV ASGV ApMV PNRSV PPV

1. Bright and Early P1 + - - - - -

2. Crytorian P1 + - + - - -

3. Gala P1, P3 + - - + - -

4. Gold Spur P1 + - - - - -

5. Golden Delicious P1 + - - - - -

6. Golden Hornet P1 + - - - - -

7. Oregon Spur P1, P3 - - - - - -

8. Red Chief P1-3 + + + + - -

9. Red Fuji P1,P3 + - + - - -

10. Red Spur P1, P3 - - - - - -

11. Royal Delicious P1, P3 + + - - - -

12. Scarlet Gala P, P3 + - + + - -

13. Top Red P1, P2 + - - - - -

14. Silver Spur P1, P3 - - - - - -

15. Spartan P1, P3 - - - - - -

16. Starkrimson P1, P3 + - + + - -

17. Vance Delicious P1, P3 + + + - - -

18. Well Spur P1, P3 - - - - - -

Rootstocks

19. Al-narp P1, P3 + - - -

20. MM111 P1- 3 + - - - - -

21. B-9 P1, P3 + - - - - -

22. MM106 P1- 4 + - - - - -

23. M9 P1, P3 + - - + - -

24. M4 P1, P3 - - - - - -

25. M7 P1- 4 + - - - - -

26. M26 P1 + - - - - -

27. M793 P3 - - - - - -

- = ELISA reading less than or equal to negative control

Results

127

Table 4.4: ELISA result for apple and other pome fruits from various locations.

S.No. Cultiva

r District

ELISA

ACLSV ASGV ASPV ApMV PNRSV PPV

1. Gala

Kullu (3) + 3 - - - - -

Kangra (5) + 5 - - - - -

Solan (5) + 5 - - - - -

Gandarbal(3

)

+ 2 - - - - -

Srinagar (5) + 3 - - - - -

2. Golden

Deliciou

s

Kullu (3) + 3 - - - +

Kinnaur (3) + 3 - - - - -

Kangra (5) + 5 - - - - -

Mandi (3) + 2 - - + -

Shimla (3) + 2 + - - + -

Pulwama (3) + 1 - - - - -

Srinagar (5) + 2 - - - - -

3. Top Red

Solan (5) + 5 - - - - -

Kullu (3) + 2 - - - - -

Kangra (5) + 5 - - - - -

Shimla (3) + 2 - - - - -

Srinagar (5) + 2 - - - - -

4. Vance

Deliciou

s

Kullu (3) + 2 - - - - -

Kangra (5) + 5 + - - - -

Solan (5) + 5 - - - - -

Pulwama (3) - - - - - -

Srinagar (5) + 2 - - - - -

5. Royal

Deliciou

s

HP (other

districts)(25) + 24 - - - - -

Mandi (3) + 2 - - + - -

Shimla (3) + 2 + - - - -

Kangra (5) + 4 + - - - -

Gandarbal(3) + 2 - - - - -

Srinagar (5) + 3 - - - - -

Pulwama (3) - - - - - -

Contd

…

Results

128

S.No

. Cultivar Location

ELISA


6. Red Gold

Kullu (3) + 3 - - - - -

Kangra

(5) + 5 - - - - -

Solan (5) + 4 - - - - -

Kullu (3) + 2 - - - - -

Shimla (3) + 3 + - - - -

Srinagar

(5) + 3 - - - - -

7. Starkrimso

n

Kullu (3) + 3 - - - - -

Kangra

(5) + 5 + - + - -

Solan (5) + 5 - - - - -

Srinagar

(5) + 3 - - - - -

8. Well Spur

Kullu (3) + 2 - - - - -

Shimla (3) + 3 - - - - -

Kangra

(3)

- - - - - -

Solan (3) + 2 - - - - -

Srinagar

(5) - - - - - -

9. Tydeman’s

Kullu (3) + 2 - - - - -

Kangra

(5) + 5 + - - - -

Solan (5) + 5 - - - - -

Shimla (3) + 3 + - - - -

Srinagar

(5)

+ 1 - - - - -

10. Scarlet

Gala

Kullu (3) + 3 - - - - -

Kangra

(5) + 5 - - - - -

Solan (5) + 5 - - - - -

Srinagar

(5) + 2 - - - - -

11. Lal Ambri Srinagar

(5) + 4 + - - - -

12. Mahi’s

Delicious

Srinagar

(5) + 2 - - - - -

Contd

…

Results

129

S.No

.

Cultiva

r Location

ELISA


13. Crytoria

n

Kullu (2) + 2 - - - - -

Kangra (5) + 5 + - - - -

Solan (3) + 3 - - - - -

Srinagar (3) - - - - - -

14. Red Spur

Kullu (3) + 2 - - - - -

Kangra (3) - - - - - -

Solan (5) + 4 - - - - -

Srinagar (5) - - - - - -

15. Red

Chief

Kullu (3) + 3 - - - - -

Kangra (5) + 5 + + - - -

Shimla (3) + 3 - - - - -

Solan (5) + 5 - - - - -

Gandarbal

(4)

+ 1 - - - - -

Srinagar (5) + 2 - - - - -

16. Richard Shimla (2) + 1 + + - -

17. Firdos Pulwama (3) - - - - - -

Srinagar (5) - - - - -

18. Spartan Srinagar (5) - - - - - -

19. Shirin Srinagar (5) + 1 - - - - -

20. Golden

Spur Shimla (3) + 3 + - - - -

21. Richi-a-

Red Manali (2) - - - + - -

22. Red Fuji

Kullu (3) + 3 - - - -

Kangra (5) + 5 + - - - -

Solan (5) + 5 - - - - -

Gandarbal

(5)

+ 1 - - - - -

Srinagar (5) + 1 - - - - -

23. Red

Deliciou

s

Shimla (4) + 4 - - + - -

Contd

…

Results

130

+ = atleast one sample from all three/five samples tested were positive (>2times –ve

control), while number of positive samples have been indicated for ACLSV

- = ELISA reading less than or equal to negative control

S.No

.

Cultiva

r Location

ELISA


24. Silver

Spur

Kullu (3) + 2 - - - - -

Kangra (4) - - - - - -

Solan (5) - - - - - -

CITH (5) - - - - - -

25. Gala

Mast

Shimla (3) + 3 + - - - -

Srinagar (5) + 4 - - - - -

Pulwama

(5)

+ 4 - - - - -

26. Keseri Shimla (2) + 2 - - - - -

27. Snow Drift Shimla (2) - - - - - -

28. Gloster Shimla (2) - - - - - -

29. Manchuri

an Shimla (3) - - - - - -

30. Red

Flush

Shimla (2) - - - - - -

31. Golden

Hornet Shimla (3) - - - - - -

Other pome fruits

32. Pear

Kangra

(15)

+ 6 + - - - -

Kullu

(5) + 1 - - - - -

Solan (5) + 2 - - - - -

Salooni

(5) + 2 - - - - -

Jammu

(5)

- - - - - -

33. Quince

Kangra

(4) + 2 - - - - -

Kullu

(3) - - - - - -

Salooni

(3) + 2 - - - - -

Solan (3) - - - - - -

Srinagar

(2) - - - - - -

Results

131

negative control) the cultivar was considered to be ACLSV infected. Survey of established

orchards in HP and J&K indicated towards high incidence of ACLSV infection on various

apple cultivars. From surveys in HP it was evident that a significant percent of other pome

fruits were also ACLSV positive. Quince samples from Salooni and Palampur showed

symptoms of leaf chlorosis while, pear which were apparently symptomless tested positive

from several locations. Limited surveys in J&K pointed towards ~32.6% ACLSV incidence

on apple. Among other viruses ASPV followed by ASGV were major viruses on apples.

While, ApMV was more common in cultivars from J&K in comparison to cultivars from HP.

ACLSV came across as a major virus on apple with disease incidence of ~80.8% in HP alone.

Percent disease incidence of apple and other pome fruits is listed in Table 4.4. A total of 290

apple plants were tested throughout the study and of these 90 were symptomatic while 200

were apparently healthy. The percent disease incidence of ACLSV on apple in HP and J&K

was ~75 %. The results of only spring season were evaluated to calculate disease incidence as

ELISA readings during autumn were ambiguous and unreliable. Most of the symptomless

plants also tested positive by ELISA.

4.1.2.2 Cherry

Cherry orchards mainly in J&K and Shimla district of HP were surveyed. Limited number of

cherry trees from mixed plantations at Kangra, Kullu and Solan were also indexed.

Commercial cultivars (Table 4.4) were mostly symptomless in HP areas. While mosaic,

puckering, fruit deformations, bronzing and leaf enations were common in cultivars from

J&K. Cultivars from J&K viz. Mishri and a few samples from HP gave mild reaction with

ACLSV antibodies. Out of 60 samples tested by ELISA 25 (41.7%) were positive for

ACLSV. A survey was also undertaken to identify viruses infecting naturally growing wild

Himalayan cherry in the major fruit growing and adjoining forest areas (Kullu, Solan, Shimla

and Kangra) of the state of HP, India. Some of the trees showed mild symptoms, although

most of the plants were apparently healthy. Forty samples were collected from these locations

and each sample included 8-10 leaves per tree. Positive reactions were obtained for 32.5%

(13 plants) samples. The strongest positive reaction was obtained in a sample from the

Kangra area. Among other viruses tested cultivar Lupin (J&K) was positive for PNRSV and

PPV. ASGV, ApMV and ASPV were also detected in many commercial cultivars.

4.1.2.4 Apricot

Results

132

About 53 apricot plants (30 from cultivated and 23 from wild) were screened by ELISA to

detect ACLSV and other viruses. Only 14 (26.6%) samples were ACLSV positive. Apricot

(New castle, Hard core and Early Grandson) from Palampur area was positive for both

PNRSV and ApMV (Table 4.5).

4.1.2.3 Almond

Almond is not grown on large scale in HP but, is an important cash crop of J&K. In HP,

almonds are grown in mixed plantations with pome and other stone fruits and, therefore, may

be exposed to the viruses that more commonly infect these plants. Representative samples

(20) from 6 locations in were tested by ELISA for ACLSV and other apple viruses. Leaf

samples from Bajaura, Nauni, Palampur, Salooni, Tissa and Attari area of states of HP and

Punjab were collected. There were no visible symptoms observed and plants looked

apparently healthy. Only one sample from Palampur was positive for ACLSV while

4.1.2.5 Peach / Nectarines

Peach samples (60) from the states of HP (Kullu, Mandi, Solan, Shimla, Kinnaur and

Kangra), J&K (Srinagar) and Punjab (Amritsar) were indexed to identify the incidence of

ACLSV infection in peach plantations. Mild chlorotic ring-like symptoms, necrotic spot and

shot holes were commonly observed on some leaves; however most of the plants were

apparently healthy and no disease symptoms were recorded. A total of 13 orchards were

surveyed. From each orchard 5-10 different trees were selected at random and three samples

of leaves were taken from each tree. Mixed infection of ACLSV and PNRSV was shown by

ELISA results of peach cultivar Elberta from Kullu and another cultivar Shan-e-Punjab from

Palampur was found infected by ACLSV alone. In almost all orchards checked for presence

of the viruses, these two varieties were found infected. For nectarines 3-5 plants from every

orchard were analyzed however, none of the nectarine samples were positive

Results

133

Table 4.4: ELISA results for different viruses infecting cherry

S.No. Cultivar Location ELISA


1. Stella (10) Srinagar + + +

2. Mishri (10) Srinagar + (7) + + +

3. Lupin (10) Srinagar + +

4. Manigam (10) Srinagar + (3)

5. Sunburst (10) Srinagar

6. Makhmali (10) Srinagar

7. Cherry (5) Kullu + (3) +

8. Cherry (5) Kangra + (5)

9. Cherry (10) Solan

10. Cherry Red

(20) Shimla + (7) +

+ Sign indicates positive results in ELISA and blank spaces indicate negative result

Table 4.5: ELISA results for different viruses infecting apricot

S.No. Cultivar Location ELISA

ACLSV ASPV ASGV ApMV PNRSV PPV

1. New

castle

Kangra + + +

Kullu +

Jammu

Attari

2. Hard core Kangra +

Kullu

Jammu

3. Florida

prince

Kangra +

Kullu

Nauni

Jammu

4. Early

Grandson

Kangra +

Kullu

Jammu


Results

134

Table 4.6: Incidence of different viruses infecting almond by ELISA

S.No

. Cultivar

Locatio

n

ELISA

ACLS

V

ASP

V

ASG

V

ApM

V

PNRS

V PPV

1. Prabhat Jammu

2. Mukdhu

m Jammu

3. Shalimar Jammu

4. Waris Jammu

5. Almond HP + +

+ sign indicates positive results in ELISA and blank spaces indicate negative result

Table 4.7: Incidence of different viruses infecting peach/nectarine by ELISA

S.

No.

Cultiva

r

Locatio

n

ELISA

ACLS

V ASGV ASPV ApM

V

PNRS

V

PP

V

1. Elberta

Kullu + +

Kangra

Solan

Jammu

2. Shan-e

Punjab

Kangra +

Amritsar

Solan

Jammu +

3. Florida

Sun

Kullu

+

Kangra

Solan

Jammu

4. May

Fire

Kullu +

Kangra +

Shimla +

Solan +

5. Snow

Queen

Kullu

Shimla +

Kangra +

Solan


Results

135

for ACLSV. Nectarine cultivars May Fire and Snow Queen from Palampur, Solan and Kullu

areas were found infected by PNRSV (Table 4.7).

4.1.2.5 Plum

Among other natural hosts of ACLSV plums from HP, J&K, Punjab were analysed by ELISA

to estimate the disease incidence of ACLSV on plum. About 26% (13/50 samples) tested

were ACLSV positive. ApMV and PNRSV infection was also very widespread in plum

cultivars.

4.1.3 Standardization of Diagnostics

4.1.3.1 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

RT-PCR was done for confirming the presence of ACLSV on apple, other pome and stone

fruits using ACLSV specific primers at already standardized conditions (Menzel et al., 2002)

for the detection which amplified approximately 675 bp DNA fragments containing partial

CP gene. RT-PCR for other apple viruses/viroid(s) was also attempted at given conditions

(Menzel et al., 2002; Choi et al., 2003; Mackenzie et al., 1997; Kulshrestha, 2005 Faggioli

and Ragozzino, 2002). ELISA positive samples with maximum and borderline absorbance

values were tested. Total RNA extracts of these samples were obtained using commercial kits

and/or classical methods and used for RT followed by PCR. RT-PCR was also done for

ASPV, ASGV, ApMV, PNRSV, PPV and ASSVd using specific primers. Required

amplifications were obtained for all except PPV. Gel photographs showing desired

amplification are shown in Fig 4.2. All the desired amplifications obtained were cut, eluted,

ligated into a pGEM-T easy cloning vector and transformed using E. coli DH5 strain

competent cells with ampicillin selection. Positive clones were identified after restriction

digestion and pure plasmid from positive clones was sequenced by an automated sequencer

using universal primers. The sequences obtained on BLAST analysis showed sequence

identity to desired pathogens. However, sequenced amplicon of ASPV was found to be a non-

specific. All the confirmed sequences were submitted to EMBL database (Table 4.9).

4.1.3.1 Slot Blot Hybridization for Diagnosis

Plant samples were also analyzed by hybridization, which is a more sensitive

Results

136

Table 4.8: Incidence of different viruses infecting plum by ELISA

S.

No.

Cultivar Location ELISA


1. Kala

Amritsari

Kangra +

Kullu

Jammu

Amritsar + +

2.

Santa

Rosa

Kullu

Chamba

Kangra + +

Solan +

Shimla +

Jammu +

Amritsar

3. Frontier Kullu +

Solan

Kangra

Jammu +

4. Mariposa Kullu

Solan

Kangra + +

Jammu

+ sign indicates positive results in ELISA and blank spaces indicate negative result

Results

137

technique compared to ELISA. Already sequenced ACLSV CP from apple was labeled with

α32

-P dATP and used as probe in hybridization. Apart from the samples found positive by

ELISA some new samples also tested positive by hybridization (Fig 4.3). This technique

further confirmed the presence of the ACLSV in pome and stone fruits. Positive results were

found in apple (Scarlet Gala from CITH, Srinagar), Almond (Solan) and Cherry (Shimla).

Similarly more positive samples were identified by using radiolabelled probes of ASGV,

ApMV and PNRSV (Table 4.10).

4.2 VIRUS TRANSMISSION AND HOST RANGE

4.2.1 Virus Transmission

For the transmission of ACLSV, some plant samples showing maximum and borderline

ELISA values were selected. The samples were macerated using sterile pestle and mortar in

0.01M sodium phosphate buffer, pH 7 containing 2% PVP (M. wt. 8000) (Lister, 1970).

4.2.1.1 Mechanical Transmission of ACLSV

Healthy plants (4-8 leaf stage), pre-dusted with carborandum, of diagnostically susceptible

host species Chenopodium amaranticolor, C. quinoa, Vigna sinensis (var. Chitlidana),

Phaseolus vulgaris and diagnostically insusceptible host species (3-5 leaf stage) Nicotiana

glutinosa and Cucumis sativus (cotyledons) were inoculated as described earlier. After 8-12

days post inoculation necrotic and/or chlorotic local lesions appeared on inoculated leaves

followed by systemic chlorosis and leaf deformations indicating that the virus transmitted

mechanically. Mechanical inoculation with ACLSV India11 isolate (apple, Kinnaur) showed

severe leaf deformation, vein clearing and chlorosis on C. amaranticolor, severe chlorosis on

C. quinoa; severe mottling, chlorosis and necrotic spots on P. vulgaris and severe necrotic

lesions on V. sinensis (var. Chitlidana) (Fig 4.6 A).

Mild chlorosis on C. amaranticolor and mild necrotic lesions on V. sinensis (var. Chitlidana)

were obtained by inoculating ACLSV isolate India13 (apple, Palampur). Severe chlorosis and

curling on C. amaranticolor; necrotic spots on V. sinensis (var. Chitlidana); severe chlorosis

on C. quinoa and mild chlorotic spots on P. vulgaris was observed with India 15 (apple,

Kashmir) isolate. India16 (apple, Uttarakhand) isolate showed severe chlorosis and vein

clearing on V. sinensis (var. Chitlidana)

Results

138

Fig 4.2 : Gel photographs showing desired amplicons obtained by RT-PCR using detection

primers from randomly selected ELISA positive samples (A) ACLSV: ~ 677 bp amplicons

and 1kb ladder, (B) ASGV: ~755bp amplicons, (C) ApMV: ~700 bp amplicons, (D) PNRSV:

~675bp amplicons, (E) ASPV: ~370 bp amplicon, (F) ASSVd: ~300 bp amplification of

complete genome. All other gels (B-F) show 100 bp marker.

marker.

Fig 4.5:

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 23 24

Fig 4.5: Autoradiograph for detection of ACLSV by hybridization. Slot 1 and 22-positive

controls, slot 2- blank, slot 21- negative control, slots 3 to 18- 16 apple cultivars, slot 19, 23,

24 apple rootstocks MM106, B-9, M7 respectively.

Results

139

Table 4.9: Accession numbers of the sequences submitted to the EMBL databank.

Table 4.10: List of samples that tested positive by Hybridization

S.No. Pathogen Cultivar

(Host / location) Accession numbers

1. ACLSV Royal Delicious apple

from Palampur, Solan

AM409323, AM409324-

Palampur, AM409321-Solan,

2. ASGV Starkrimson apple

from Shimla FM204881

3. ApMV Golden Delicious

apple from Shimla

FM178274, FJ429311,

FJ429309

4. PNRSV

Golden Delicious

apple from Kullu,

Kalpa

AM419814-Kullu and

AM491772-Kalpa

5. ASSVd Starkrimson apple

from Shimla

AM993159, AM993160,

FM178283, FM178284,

FM178285, FM208138,

FM208139, FM208140,

FM208141, FM208142

S.No. Pathogen Cultivar

(Host / location) ELISA Hybridization

1. ACLSV

Scarlet Gala (apple,

Srinagar) -ve +ve

Gala (apple, Kullu) -ve +ve

Almond (Nauni) -ve +ve

Cherry (Srinagar) -ve +ve

Cherry (Shimla) -ve +ve

2. ASGV Cherry (Solan) -ve +ve

3. ApMV Golden Delicious (Shimla) -ve +ve

4. PNRSV Almond (Nauni) -ve +ve

Results

140

(Fig 4.7 A, B, C) while India20 (peach, Bajaura) showed severe chlorosis on C.

amaranticolor and C. quinoa. ACLSV India27 isolate (wild Himalayan cherry, Palampur)

showed severe leaf curling and chlorosis on V. sinensis (var. Chitlidana) and severe chlorotic

spotting on C. amaranticolor. However, only mild chlorosis was obtained on C. quinoa while

P. vulgaris developed mild chlorotic spots and mottling (Table 4.10, Fig 4.6 B).

4.2.2 Host Range and Symptomatology

Host range was carried out by mechanical inoculations on different host plants. For this,

extract was prepared from young infected leaves of C. quinoa singly infected with ACLSV

maintaining pure culture. The results of host range are summarized in Table 4.11. Sixteen

plant species belonging to six families were used for host range studies. Of these 16 plant

species only five were found to be susceptible for ACLSV infection. After back inoculation

from these plants local lesions were produced on C. quinoa and/ or P. vulgaris. Few ACLSV

isolates also showed chlorosis on some of the Nicotiana spp. (Fig 4.8). All the symptomatic

N. benthamiana and N. megalosiphon plants were ELISA positive for ACLSV. However,

none of them tested positive by RT-PCR.

4.2.3 Maintenance of Virus Culture

The culture of ACLSV used in this study was obtained from apple and wild Himalayan

cherry. The pure culture was maintained both in vivo and in vitro.

4.2.3.1 In vivo

On the basis of differential host range, pure virus culture for apple cultivar Gala Mast from

Gandarbal (J&K), Gala from Uttarakhand and Wild Himalayan cherry (inoculation at 4-8 leaf

stage) and P. vulgaris (inoculation at cotyledonary leaf stage). To ensure the biological purity

of the virus and to eliminate possible contamination from other viruses, the pure culture was

developed on the leaves of C. quinoa /C. amaranticolor /P. vulgaris and back inoculating of

the ACLSV infected host sap diluted with 0.01M sodium phosphate buffer having pH 7

containing 2% PVP (M. wt. 8000). Purity of the virus culture was checked periodically by

back inoculation on C. quinoa / P. vulgaris and reconfirmation at molecular level as seen

Results

141

Table 4.10: Details of symptoms obtained on various herbaceous plants after mechanical

inoculation.

“- “ no symptoms.

S.No

. Sample

C.

amaranticolor

C.

quinoa

V. sinensis

(var.

Chitlidana)

P. vulgaris

1.

India11

(apple,

Kinnaur)

Severe leaf

deformation,

vein clearing

and chlorosis

Severe

chlorosis Necrotic lesions

Severe mottling,

chlorosis and

necrotic spots

2.

India27

(wild

Himalayan

cherry,

Palampur)

Severe

chlorotic

spotting

Mild

chlorosis

Severe leaf

curling and

chlorosis

Mild chlorotic

spots and mottling

3.

India13

(apple,

Palampur)

Mild chlorosis - Mild necrotic

lesions Mild chlorosis

4. India 15

(apple,

Kashmir)

Severe curling

and leaf

deformation

Severe

chlorosis Necrotic spots Mild chlorosis

5.

India16

(apple,

Uttarakhand)

Mild chlorosis - Severe chlorosis

and vein clearing -

6.

India 20

(peach,

Bajaura)

Severe

chlorosis

Severe

chlorosis - -

Results

142

(A)

(B)

Fig 4.6: Symptoms on various susceptible hosts after mechanical inoculation of India11

isolate (A) and (B) India27 isolate.

Results

143

(A)

(B)

(C)

Fig 4.7: Symptoms on various susceptible hosts after mechanical inoculation of (A)

India13, (B) India15 and (C) India16 isolate.

Results

144

Fig 4.8: Symptoms on Nicotiana spp. after mechanical inoculation of various samples- (A)

Chlorosis on N. megalosiphon due to India11 isolate (B) Chlorotic spots and severe chlorosis

on N. benthamiana with India27 isolate

(A) (B)

Fig 4.9: (A) Lane 1, 2, 3 are desired amplicons (~850bp) of CP from Gala Mast from

Gandarbal (J&K), Gala from Uttarakhand and wild Himalayan cherry from Palampur (HP,

maintained in vivo after back inoculation on C. quinoa) respectively, obtained by RT-PCR

using specifically designed primers. (B) RT-PCR results for Gala from Uttarakhand (in vitro)

isolate, lanes 1-4 are negative results for ApMV, ASGV, PNRSV, ASPV amplifications, lane

5 is desired amplicons for CP Lane M is 100bp DNA ladder.

Results

145

Table 4.11: Host range of Apple chlorotic leaf spot virus as identified during present

studies

(-) No symptoms were obtained.

S.No. Family Host Plants Symptoms

1. Amaranthaceae Amatanthus caudatus -

2. Caryophyllaceae Dianthus barbatus -

3. Chenopodiaceae Chenopodium quinoa Chlorosis

C. amaranticolor Chlorosis, vein clearing and

leaf deformation

Gompherena globosa -

4. Cucurbitaceae Cucumis sativus -

5. Fabaceae Cymopsis

tetragonoloba

-

Phaseolus vulgaris mottling, chlorosis and

necrotic spots

Vigna sinensis Necrotic lesions, chlorosis

and leaf deformation

6. Solanaceae Lycopersicon

esculantum

-

Nicotiana benthamiana Mild mosaic

N. clevelandii -

N. glutinosa -

N. megalosiphon Mosaic and mottling

N. rustica -

N. tabacum -

Results

146

in (Fig 4.9 A).

4.2.3.2 In vitro

To maintain pure culture of the virus in vitro tissue culture was used. ACLSV infected apple

cultivar Gala from Uttarakhand was first of all tested for the presence of other apple viruses

such as ApMV, ASPV, ASGV and PNRSV by ELISA and RT-PCR using virus specific

primers (Table 3.2). The explants were found infected only by ACLSV (Fig 4.9). Infected

explants (apical/lateral meristems 0.2-1.0mm) were taken and cultured on Murashighe and

Skoog (MS) medium (Table 3.2) with 1mg/ml BAP, 0.1mg/ml NAA, 3% sucrose and 0.7%

agar. The explants were routinely subcultured at 6 weeks interval to maintain the stock and

for further use in molecular studies (Fig 4.10).

4.3 PURIFICATION AND SEROLOGICAL CHARACTERIZATION OF ACLSV

4.3.1 UV Absorption Spectrum

ACLSV purification was performed using established protocols as described in material and

methods section with minor modification. Crude virus extract (CVE) from the systemically

infected C. quinoa plants (inoculated with extracts of leaves showing chlorotic leaf spots

symptoms from Gala Mast cultivar (J&K; India15), 10-15 days post inoculation),

shoots/leaves from in vitro maintained pure culture (Gala from Uttarakhand; India16) and

young buds and flower petals (wild Himalayan cherry from HP; India27) was obtained. The

purified preparation was examined in UV spectrophotometer. Spectrum of partially purified

virus preparation showed absorption maxima at 260nm wavelength (Amax). The Absorption

ratio at A260/280 (A260/280) of crude virus preparation was calculated to be 1.5, 1.53 and 1.43

for India15, India27 and India16 isolates respectively.

4.3.2 Electron Microscopy

The crude virus extract prepared from wild Himalayan cherry flowers and Gala Mast leaves

was examined under electron microscope at 80kV and 21,000X magnification. Virion size

was determined by plotting a size distribution frequency curve of 100 virions. Most of the

virions were found to aggregate in end to end manner.

Results

147

Fig 4.10: (A) In vitro maintained ACLSV pure culture on cultivar Gala from Uttarakhand,

showing symptoms of severe chlorosis. (B) Infected shoot explants harvested from

maintained pure culture for RNA extraction.

(A) (B)

Fig 4.11: SDS-PAGE (12%) showing protein band of partially purified ACLSV preparation

(~26 kDa). (A) Lane 1, 2, 3 are preparation obtained from back inoculated C. quinoa (Gala

Mast leaves; J&K), from leaves of in vitro maintained ACLSV pure culture on Gala

(Uttarakhand) and flowers of wild Himalayan cherry (HP) respectively. (B) X-ray sheet

showing the captured luminescence, of partially purified ACLSV preparation, obtained by

western blotting. Lane M: pre stained rainbow marker (Fermentas, Lithuania).

Results

148

4.3.3 DAS-ELISA

Virus specific antibodies of ACLSV (Bioreba, USA) were used to determine the optimum

dilution of purified virus preparation of ACLSV in DAS-ELISA. Partially purified virus

reacted up to 1:500 dilutions (1:1000, 1:700, 1:500, 1:300) for ACLSV. The absorbance at

405nm for the optimum dilution of CVE of ACLSV preparations was 1.55-1.58, while the

absorbance values of ACLSV positive and negative controls were 1.70 and 0.154,

respectively.

4.3.4 SDS-PAGE of the Partially Purified Virus Preparation

Partially purified virus preparations were electrophoresed on 12% SDS-PAGE. Silver stained

gels showed single bands of approximate size 26 kDa corresponding to coat protein of

ACLSV (Fig 4.11).

4.3.5 Western Blotting

Western blots were prepared by running two similar SDS-PAGE gels and then using one gel

to transfer the protein bands of the virus to PVDF membrane. It was then incubated with

commercial ACLSV specific antibodies. The blot was then probed by anti-rabbit HRP tagged

secondary antibodies and finally developed using luminal as mentioned earlier. An X-ray

sheet exposed to the membrane captured the luminescence generated. The protein species

which were seen on the gel were specifically identified as CP. The other gel was silver

stained to visualize and confirm the presence and size of CP (Fig 4.11).

4.4 MOLECULAR CHARACTERIZATION OF ACLSV

For the characterization of ACLSV at molecular level RNA isolated from various samples by

using RNA extraction buffer along with the commercial RNA extraction kit as detailed

earlier. This RNA was used in RT-PCR to amplify coat protein (CP), movement protein (MP)

and replicase using indigenously designed gene specific primer pairs. The PCR conditions

were standardized and desired amplicons were cloned, sequenced and analysed with already

established sequences in the database.

4.4.1 Extraction of Viral Nucleic Acid

The viral nucleic acid was isolated from original host leaves/ flowers and inoculated

herbaceous hosts along with the plant RNA. The concentration of nucleic acid was

calculated by using the formula given below:

40 x A260 x dilution factor = [µg/ml]

Results

149

The concentrations of the total RNA extracted using CTAB method and Qiagen kit ranged

approximately 0.9-1.5 and 0.2-0.4 µg/µl respectively for various samples.

4.4.2 Primer Designing

Various primer pairs were designed for the amplification of different genes of ACLSV (Table

4.12). The gene sequence of primer pairs were submitted to EMBL database. Bold underlined

sequences in ACLSV CP up (U) and down (D) primers are restriction enzyme site of Bam HI

and Hind III respectively designed for in-frame cloning in expression vector pET 32a ( Table

4.13).

4.4.3 Standardization of RT-PCR for complete CP, MP and Replicase

RT reaction was performed as previously described using gene specific down primer. PCR

amplification of different genes of ACLSV was standardized for various hosts at various

temperatures and magnesium concentrations and at different PCR conditions using Taq DNA

polymerase (Genei, India) for smaller fragments and Protoscript® AMV Long Amp

TM Taq

RT-PCR Kit (USA) for the amplification of longer fragments. The patterns of thermal cycling

for the amplification that gave the best results of various fragment is summarized in Table

4.14. Agarose gel electrophoresis of the PCR products revealed desired amplicons (Fig 4.12)

corresponding to complete CP, MP, parts of replicase and various ACLSV genome parts.

There was no amplification in healthy C. quinoa sample used with the above RT-PCR

reactions as negative control.

4.4.4 Cloning of PCR Amplified Products and Checking of Clones

The amplified fragments of ACLSV CP, MP parts of replicase were eluted from the gel,

ligated into pGEM-T easy vector system (Promega). E.coli strain DH 5α was transformed

with the recombinant plasmid and selected on Luria agar ampicillin plates. The transformed

clones were checked for the presence of gene of interest by restriction digestion. Restriction

digestion analysis (with Eco RI/ Pvu II) gave desired length of insert in positive clones.

Results

150

Table 4.12: Designed Primers used genome characterization of Indian strain of ACLSV

Table 4.13: Sequence of expression primers used

Name Primer sequence in 5’ to 3’ orientation

ACLSV CP U GGATCC ATG GGG GCA GTG CT

D AAGCTT TTA AAC GCA AAG ATC AGT T

Region Primer sequence in 5’ to 3’ orientation Product

size

Accession

Number

28-1583

up CGT GAG TAA ACA GAT TGA CGTA

~1.5kb

FN869118

dn GAC GTG GAT GAC AGC AAA TCA GA

submitted

4502-4922

up GTC AAT CAA GCG ATG AGT TAT GAG GC

~420bp

-do-

dn GTG TAT CGA CAC CAT GGG CTA AAT TC

-do-

5700-7106

complete

MP

up TTG AGT GG(C/T) T(T/G)T C(A/T/G)G ACG

~1.4kb

-do-

dn GC(C/T) TCA CA(C/C/G) AC(C/T) TG(G/C/A) CGG AA

-do-

6766-7552

complete CP

up GAT CAG AAG A/GA/CG G/AAG GAT ~786bp

AM490253

dn GTA GTA AAA TAT TTA AAA G AM490254

5700-6112

up TTG AGT GG(C/T) T(T/G)T C(A/T/G)G

ACG ~412

submitted

dn GTC ACC AAG GTG GCG GAA CCA

CTA TC -do-

Results

151

Table 4.14: Thermal cycling pattern for amplicons obtained for Indian strain of ACLSV

Region Thermal Cycling Pattern Taq used

1-1583

Replicase

Initial denaturation for 30sec at 95°C and 45 cycles

consisting of 94°C for 30sec, annealing temperature of

58°C for 30sec and elongation at 68°C for 1.55min . Final

elongation for 5min at 68°C.

Protoscript

5700-7106

complete MP

Same as above with annealing at 52°C for 30sec followed

by elongation at 68°C for 1.5min.

Protoscript

4502-4922

Replicase

Initial denaturation for 2min at 95°C and 32 cycles

consisting of 94°C for 1min, annealing temperature of 64°C

for 1min and elongation at 72°C for 1min . Final elongation

for 10min at 72°C.

Genei

6766-7552

complete CP

Same as above with annealing at 47-50°C for 1min followed

by elongation at 72°C for 2min.

Genei

5700-6112

Replicase/MP

Same as above with annealing at 58°C for 1min followed by

elongation at 72°C for 2min.

Genei

Fig 4.12: 1% Agarose gel stained with ethidium bromide showing various amplicons of

ACLSV genome from Gala Mast (J&K) isolate (A) lane1: complete CP ( ~800bp) (B) lane 1:

~1580 bp sequence of replicase

Results

152

4.4.5 Sequencing

The plasmid DNA containing the gene of interest was purified using GenElute Plasmid

isolation kit (Sigma, India) and three clones of each gene were sequenced and consensus

sequences were taken for further analysis using various bioinformatics tools. Sequencing

revealed that CP amplicons (~800bp) gave 584bp (193aa) CP and 3’end un-translated region

(UTR). Twenty-nine isolates of CP were characterized from various hosts and locations

(Table 4.15). About 1405bp of MP amplicons had 1386 bp (460aa) long gene as expected.

Two partial 5’-end MP sequences (~141aa) from HP (Palampur and Kinnaur) were also

sequenced (Table 4.16). Apart from this deduced amino acid sequence of 3’-end of ACLSV-

MP (~110aa), overlapping with the CP was deduced by frame shift reading of Indian CP

isolates (Table 4.20). Replicase was amplified in two parts using J&K isolate of ACLSV

(India15). Sequencing revealed that first part was 1572bp (from 5’- 28nt to 1580nt -3’) and

the second 420bp (from 5’- 4502nt to 4922nt - 3’). Similarly the 3’-end region of replicase

overlapping with MP (~36aa) was obtained from Gandarbal (India15), Kinnaur (India11) and

Palampur (Red Chief) isolates (Table 4.24).

4.5 ASSESSMENT OF DIVERSITY IN THE GENOME OF ACLSV INFECTING

POME AND STONE FRUIT

The sequences of ACLSV CP, MP and replicase were analyzed using various bioinformatics

tools with corresponding sequences of other established ACLSV sequences from the database

to characterize the Indian isolate of ACLSV.

4.5.1 Phylogenetic Analysis of ACLSV Coat Protein (CP) gene Sequences

Complete sequence of ACLSV coat protein gene of about twenty-nine isolates from India

isolates were obtained from various pome (apple, pear, quince) and stone (plum, peach,

apricot, almond, Wild Himalayan cherry) fruit trees. ACLSV coat protein sequences from the

present study and those from earlier deposited sequences (partial and complete) in the

database (Table 4.15, 4.17) were subjected to multiple sequence alignment and phylogenetic

analysis.

4.5.1.1 Apple

The percent sequence identity of forty-one apple isolates, (16 Indian characterized in this

study [91-100% identity at aa level] and 25 others) at amino acid (aa) level

Results

153

Table 4.15: All the Indian ACLSV-CP isolates characterized from India

S. no. Accession

no. Place Isolate CP Source Cultivar

1. AM494505 Nagri India1 Full (F) apple (Ap) Royal Delicious

2. AM494506 Dobi India2 F apple (Ap) Royal Delicious

3. AM494507 Solan India3 F apple (Ap) Royal Delicious

4. AM494508 Nihari India4 F apple (Ap) Royal Delicious

5. AM494509 Kalpa India5 F apple (Ap) Royal Delicious

6. AM494510 Bajaura India6 F apple (Ap) Golden Delicious

7. AM494511 Tissa India7 F apple (Ap) Royal Delicious

8. AM494512 Sangla India8 F apple (Ap) Royal Delicious

9. AM494513 Salooni India9 F apple (Ap) Royal Delicious

10. AM494514 Palampur India10 F apple (Ap) Vance Delicious

11. AM408891 Kinnaur India11 F apple (Ap) Royal Delicious

12. AM409322 Kotgarh India12 F apple (Ap) Red Gold

13. AM709776 Palampur India13 F apple (Ap) Bright n Early

(BE)

14. AM709777 Palampur India14 F apple (Ap) Scarlet Gala(SG)

15. FN550875 Kashmir India15 F apple (Ap) Gala Mast

16. FN550876 Uttarakhand India16 P (partial) apple (Ap) -

17. AM882705 Palampur India17 F Plum (Pl) -

18. AM931534 Palampur India18 F Plum (Pl) Kala Amritsari

19. AM882704 Palampur India19 F Pear (Pr) -

20. AM498047 Kullu India20 F Peach (Pe) Elberta

21. AM931533 Palampur India21 F Peach (Pe) Shane Punjab

22. AM498050 Solan India22 F Peach (Pe) -

23. AM498049 Salooni India23 F quince (Qu) -

24. AM498046 Solan India24 F almond (Ald) -

25. AM498045 Solan India25 F apricot (Apr) -

26. AM498048 Kullu India26 F wild apricot

(Apr)

Chuli (rootstock)

27.

AM498044 Palampur India27 F Wild

Himalayan

Cherry (Che)

rootstock

28. FN666579 Palampur India28 F -do- -do-

29. FN666578 Palampur India29 F -do- -do-

http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&val=154623405


http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=159147409


Results

154

ranged from 89-100 %. A few CP isolates (apple) from India shared 100% sequence dentity

at nucleotide and amino acid levels. Multiple alignment of the all CP (apple) isolates showed

considerable conservation in first 36 and last 55 amino acids at N and C-terminals

respectively. However, maximum non conserved changes creating variability in amino acid

sequences were in the middle portion of the coat protein gene (Fig 4.15). Phylogenetic trees

constructed (Fig 4.14 A, B) clearly indicated formation of two groups viz. Group 1 and Group

2, based on distance in phylogeny and variation / co variation of amino acids (Table 4.18).

No region wise clustering was observed. Group 1 had more variability as compared to Group

2. The conserved residues RQ/FDF (102-103/154-156) identified as a salt bridge structure

(highlighted in green) were also present in all the CP sequences (Fig 4.13, 4.15).

4.5.1.2 Other Pome Fruits (Pear and Quince)

The CP isolate from Indian pear (India19) shared 91 % sequence identity at amino acid level

to the Kuerel pear isolate (AAT75238) from China. The CP quince isolate (India 23)

exhibited sequence identity of 84 and 89% at nucleotide and amino acid respectively with the

partial CP isolates from Greece. Sequence analysis showed that all the CP (pome fruit)

isolates from India share amino acid identity of 92-100% among themselves and 92-97%

with other complete CP (pome fruit) isolates from the world. All the Group B isolates shared

the least sequence identities and showed distinct pattern of amino acid variations / co-

variations with Group A isolates (Fig

4.16). Among all the CP isolates from pome fruits the Kuerel (pear) and MO-5 (apple)

isolates showed maximum variability at amino acid level (Fig 4.16) and also

clustered separately from most of the Group 2 isolates (Fig 4.17). Radiated phylogenetic tree

(Fig 4.17) showed clustering of the India19 (pear), Kuerel (pear, China) and quince (Greece)

CP isolates in Group 2 while the India23 (quince) isolate falls in Group 1. The clustering was

supported by the covariation / changes in amino acid positions listed in Table 4.18.

4.5.1.3 Plum

All the CP isolates from plum showed maximum variability at amino acid positions 20-98

(Fig 4.18 A). Phylogenetic tree clearly showed grouping of related isolates (Fig 4.19 A). Both

the Indian isolates (India17, 18) infecting plum were 99 and 100%

Results

155

Table 4.16: Accession numbers of sequenced genes of ACLSV

S. no. Gene Accession number

1. Partial MP Palampur isolate (5’end) AM888397

2. Partial MP Kinnaur isolate (5’end) AM888396

3. Complete MP Kashmir isolate FN673831

4. Partial replicase gene Kashmir isolate

(middle region) FN555394

5. Partial replicase gene Kashmir isolate

(5’end)

FN826784, FN826785,

FN826786

Table 4.17: List of all the ACLSV-CP isolates characterized from the world. Other

Trichovirus viz. Apricot pseudo chlorotic leaf spot virus (APCLSV), Peach mosaic virus

(PeMV) and Cherry mottle leaf virus (CMLV) CP isolates used as outgroup in the study.

S. no Accession no. Isolate name CP Country Source

1. ABL63752 BR-1 Full (F) Brazil (Brz) apple (Ap)

2. P54890 - F Japan (Jap) apple (Ap)

3. D14996 P-205 F Japan (Jap) apple (Ap)

4. AB326230 GC10j F Japan (Jap) apple (Ap)

5. AB326229 GC10h F Japan (Jap) apple (Ap)

6. AB326228 GC10f F Japan (Jap) apple (Ap)

7. AB326227 GC10c F Japan (Jap) apple (Ap)

8. AB326226 GC10a F Japan (Jap) apple (Ap)

9. AB326225 MO-5 F Japan (Jap) apple (Ap)

10. AB326224 B6 F Japan (Jap) apple (Ap)

11. AB326223 A4 F Japan (Jap) apple (Ap)

12. AAT80319 AT-43 Partial (P) Hungary (Hun) apple (Ap)

13. AAT80320 AT-49 P Hungary (Hun) apple (Ap)

14. CAE52470 M93 P Albania (Alb) apple (Ap)

15. ABG75614 SKIL P Isarel (Isr) apple (Ap)


17. CAE52468 M54 P Italy (Ita) apple (Ap)

18. ABC59575 P10R1D3 P Bulgaria (Bul) apple (Ap)

19. CAE52485 MP-Tur P Turkey (Tur) apple (Ap)

20. CAE52486 MP02 P Italy (Ita) apple (Ap)

Contd…

Results

156





24. CAE52484 MP-CI P China (Chi) apple (Ap)

25. ABK62735 ACLSV-C F China (Chi) apple(Ap),

peach(Pe)

26. AJ586650 PE154 P Hungary (Hun) peach (Pe)

27. EU223295 TaTao F USA peach (Pe)

28. AJ586651

(APCLSV) PE297 P Jordan (Jor) peach (Pe)

29. AAU93348 HBP F China (Chi) peach (Pe)

30. AJ586646 PE 118D P Hungary (Hun) peach (Pe)

31. AJ586644 PE- FC P Italy (Ita) peach (Pe)

32. AAU06132 AP 10 P Turkey (Tur) peach (Pe)

33. ABC59574 R1D2P-L P Bulgaria (Bul) peach (Pe)

34. AJ586652 PE 56 P Italy (Ita) peach (Pe)

35. AJ586649 PE153 P Lebanon (Leb) peach (Pe)



38. AJ586650

(APCLSV) PE154 P Hungary (Hun) peach (Pe)

39. AJ586645

(APCLSV) PE150 P Italy (Ita) peach (Pe)

40. AAU06131 KP2 P Turkey (Tur) peach (Pe)

41. ABC59572 R2D43 P Bulgaria (Bul) peach (Pe)

42. AAT80323 P-1 P Hungary (Hun) peach (Pe)

43. AAT75238 Kuerel F China (Chi) pear (Pr)

44. AM292923 - P Greece (Gre) quince (Qu)

45. AE52472 AlF5 P Italy (Ita) almond (Ald)

46. AJ586621 Al-19 P Italy (It) almond (Ald)

47. DQ329160 P1R9D9 P Bulgaria (Bul) sweet cherry (Che)

48. X99752 Balton-1 F France (Fra) sweet cherry (Che)

49. AY730560 ASwC43 P Turkey (Tur) sweet cherry (Che)

50. AY677105 C-1 P Hungary (Hun) wild cherry (Che)

51. AY677106 C-2 P Hungary (Hun) wild cherry (Che)

52. AAF67188 SX/2 F Poland (Pol) plum (Pl)

53. M58152 P863 F France (Fra) plum (Pl)

Contd…

Results

157


54. NC_001409 - F France (Fra) plum (Pl)

55. AJ243438 PBM1 F Germany (Ger) plum (Pl)

56. AJ586623 Apr-109 P Spain (Spa) apricot (Apr)



59. AJ586630 Apr-20 P Italy (Ita) apricot (Apr)




63. AJ586635 Apr-63 P Italy (Ita) apricot (Apr)

64. AJ586631 Apr-3 P Jordan (Jor) apricot (Apr)

65. AJ586636

(APCLSV)

APR-EA5 P Turkey (Tur) apricot (Apr)

66. ABA18642

(PeMV)

CL-2 F USA peach (Pe)

67. NP_062430

(CMLV)

SA1162-21 F USA sweet cherry (Che)

Table 4.18: Seventeen amino acid variations observed in CP of Indian isolates forming two

clusters. All the underlined amino acids show co-variation in two groups (Group 1 and Group

2) formed. The amino acids in red show co-variation having role in ACLSV infectivity

(Yaegashi et al. 2007).

Amino

acid

positions

37 40 59 60 72 75 82 83 86 88 94 97 98

Gp1 T A V L/

T V F N L Ile R P S N

Gp2 M/

I S L A

/V I Y G M A K T/

S N S

Amino acid

positions 130 137 184 192

Gp1 S G M V

Gp2 T S L/

Ile I

158

1 100

AM494505In MAAVLNLQLK VDADLKAFLA AEGRPLHGKT GAILEQTLEA IFANIAIQGT SEQTEFLDVL VEVKSMEDQK VVGSFNLKEV VNLIKIFRTT SSDPNISNMT

AM494507In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494508In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM709777In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

FN550875In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494511In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM408891In .......... .......... .......... .......... .......... .........T .......... .......... .......... ...T...S..

AM409322In .......... .......... ....S..... .......... .......... .........T .......... .......... .......... ...T...S..

AM494506In .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ...S......

AM494509In .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM709776In .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494510In .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494513In .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494512In .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM494514In .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

FN550876In .... .......... .......... .......... .......... .......... .......... ..........

101 193

AM494505In FRQVCEAFAP EARNGLVKLK YKGVFTNLFS TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKSEN EAKMSSVTTD LCI

AM494507In .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM494508In .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM709777In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

FN550875In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM494511In .......... .......... ....S..... .......... .......... .......... .......... .......... .......... ..V

AM408891In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM409322In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM494506In .......... .......... .........T ......S... .......... .......... .......... .......... .......... ..V

AM494509In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM709776In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494510In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494513In .......... .......... .........T ......S... .......... D......... .......... .......... ...L...... ...

AM494512In .......... .......... .........T ......S... .......... .......... .X........ .......... ...L...... ...

AM494514In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

FN550876In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

Fig 4.13: Multiple sequence alignment of all the Indian CP isolates (apple) using Multalin program (Corpet, 1988). Clustering in two groups,

shown as unshaded sequences (Group 1) and grey shaded (Group 2) based on the amino acid changes (highlighted in yellow and conserved

residues RQ/FDF identifying the salt bridge structure highlighted in green). Resu

lts

159

(A) (B)

Fig 4.14: Phylogenetic comparison at amino acid (aa) level for all the CP isolates from apple by Neighbor Joining method using MEGA version

4 (Tamura et al., 2007); CMLV, PeMV and APCLSV CPs are used as references. (A) Rectangular phylogenetic tree with 50% bootstrap cut-off

value. (B) Radiated phylogenetic tree using Indian isolates and only complete sequences from the world.

Group 1

Group 2

Resu

lts

160

1 100


AM494507In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494508In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM709777In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

FN550875In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494511In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AB326230Ja .......... .......... .......... .......... .......... .........Q .......... .M........ .......... .......K..

AB326223Ja .......... .......... .......... .......... .......... .........M .......... .I........ .S.....K.. ......N...

AB326227Ja .......... ...E...... .......... .......... .......... .........T .......... .I........ .S.V...K.. ......N...

AB326229Ja .......... .......... .......... .......... .......... .........T .......... .I........ .......K.. ......N...

D14996Ja .......... .......... .......... .......... .......... .......... .......... .......... .G........ .......S..

P54890Jap .......... .......... .......... .......... .......... .......... .......... .......... .G........ .......S..

AM408891In .......... .......... .......... .......... .......... .........T .......... .......... .......... ...T...S..

AM409322In .......... .......... ....S..... .......... .......... .........T .......... .......... .......... ...T...S..

AM494506In .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ...S......

AM494509In .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM709776In .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494510In .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AB326226Ja .......... .......... .......... ......M..S .......... ........LS .......... .I..Y..... ..M..A.K.. ..........

AM494513In .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AB326224Ja .......... .......... .......... ......I..S .......... ........LM .......... .I..Y..... ..M..A.K.. ..........

ABK62735Ch .......... .......... .......... .........S .......... ........LV .........E .I..Y..... ..M..A.K.. ..........

ABL63752Br .......... .......... ........R. ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. .......S..

AM494512In .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM494514In .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AB326228Ja .......... .......... .D-....... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AB326225Ja .......... ......V... .......... .....L...S .......... ........LV .......... ..N.Y...S. .D.....K.. ......NG..

CAE52482Al .... .........S .......... ........LV .........R .I..Y..... ..M..A.K.. ..........

CAE5246Alb .... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

CAE52468It .... ......I..S .......... ........LA .......... .I..Y..... ..M..A.K.S ..........

CAE52470Al .... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..G....S..

ABG75614Is .... ......I..S .......... ........LV .......... .I..Y..R.. ..M..A.K.. .......S..

CAE52485Tu .... .........S .......... ........LV .......... .I..Y..... ..M..A.K.. .......S..

FN550876In .... .......... .......... .......... .......... .......... .......... ..........

CAE52481Al .... .......... .........S .........T .......... .I........ ....E..... ..........

CAE52486It .... .......... .......... .........T .......... .I........ .......... .L....N...

CAE52484Ch .... ..T....... .......... .........T .......... .I.T...... .S.....K.. ......N...

CAE52483Ap .... .....R.... .......... .........M .......... .I........ .G........ ......N...

ABC59575Bu .........S .......... ........LV .......G.. .I..Y..... ..M..A.K.. ..G.......

AAT80320Hu .T....M.S .......... ........LV .......... .I.AY..... ..M..A.K.. ..........

AAT80319Hu .T...M..S .......... ........LA .......... .I..Y..... ..M..A.K.. ..........

Resu

lts

161

101 193


AM494507In .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM494508In .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM709777In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

FN550875In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM494511In .......... .......... ....S..... .......... .......... .......... .......... .......... .......... ..V

AB326230Ja .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AB326223Ja .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AB326227Ja .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AB326229Ja .......... .......... .......... .......E.. .......... .......... .......... .......... .......... ...

D14996Jap .......... .......... .......... .T........ .......... .......... .......... .......... .......... ..V

P54890Jap .......... .......... .......... .T........ .......... .......... .......... .......... .......... ..V

AM408891In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM409322In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM494506In .......... .......... .........T ......S... .......... .......... .......... .......... .......... ..V

AM494509In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM709776In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494510In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AB326226Ja .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494513In .......... .......... .........T ......S... .......... D......... .......... .......... ...L...... ...

AB326224Ja .......... .......... .........T ......N... .......... .......... .......... .......... ...L...... ...

ABK62735Ch .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

ABL63752Br .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494512In .......... .......... .........T ......S... .......... .......... .X........ .......... ...L...... ...

AM494514In .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AB326228Ja .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AB326225Ja .......... ...D...... .........T .......... .......... .......... .......... .......... ...L...... ...

CAE52482Al .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

CAE5246Alb .......... .......... .........T ......S... .......... .......... .......... .......... ...LP..... ...

CAE52468It .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

CAE52470Al .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

ABG75614Is .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

CAE52485Tu .......... .......... .........T ......S... .......... .......... .......L.. .......... ...L...... ...

FN550876In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

CAE52481Al .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

CAE52486It .......... .......... .......... .......... .F........ .......... .......... .......... .......... ...

CAE52484Ch .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

CAE52483Ap .......... .......... .......... .......... .......... .......... ....D..... .......... .......... ...

ABC59575Bu .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AAT80320Hu .......... .......... .........T ......S... ..........

AAT80319Hu .......... .......... .........T ......S... ..........

Fig 4.15: Multiple sequence alignment of CP isolates (apple) from world using Multalin program (Corpet, 1988). Clustering in two groups

shown as grey shaded (Group 2) and unshaded sequences (Group 1). Indian isolates (red font), aa changes (yellow) and salt bridge aa (green).

Resu

lts

162

1 100

AM498049Qu MAAVLNLQLK VDADLKAFLA AEGRPLHGKT GAILEQTLEA IFANIAIQGT SEQTEFLDVL VEVKSMEDQK VVGSFNLKEV VNLIKIFRTT SSDPNISNMT

AM494505Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494507Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494508Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM709777Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

FN550875Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494511Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AB326230Ap .......... .......... .......... .......... .......... .........Q .......... .M........ .......... .......K..

AB326223Ap .......... .......... .......... .......... .......... .........M .......... .I........ .S.....K.. ......N...

AB326227Ap .......... ...E...... .......... .......... .......... .........T .......... .I........ .S.V...K.. ......N...

AB326229Ap .......... .......... .......... .......... .......... .........T .......... .I........ .......K.. ......N...

D14996JapA .......... .......... .......... .......... .......... .......... .......... .......... .G........ .......S..

P54890ApJa .......... .......... .......... .......... .......... .......... .......... .......... .G........ .......S..

AM408891Ap .......... .......... .......... .......... .......... .........T .......... .......... .......... ...T...S..

AM409322Ap .......... .......... ....S..... .......... .......... .........T .......... .......... .......... ...T...S..

AM882704Pr .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM494514Ap .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM494512Ap .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM494509Ap .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM709776Ap .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494510Ap .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AB326226Ap .......... .......... .......... ......M..S .......... ........LS .......... .I..Y..... ..M..A.K.. ..........

AM494513Ap .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AB326224Ap .......... .......... .......... ......I..S .......... ........LM .......... .I..Y..... ..M..A.K.. ..........

ABL63752Ap .......... .......... ........R. ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. .......S..

ABK62735Ap .......... .......... .......... .........S .......... ........LV .........E .I..Y..... ..M..A.K.. ..........

AM494506Ap .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ...S......

AB326228Ap .......... .......... .D-....... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AAT75238Pr .......... .....R.... ...T...... .........S ....V..... ........MM ......D... .IS.Y...S. .D...V.... ......NG..

AB326225Ap .......... ......V... .......... .....L...S .......... ........LV .......... ..N.Y...S. .D.....K.. ......NG..

CAE52482Ap .... .........S .......... ........LV .........R .I..Y..... ..M..A.K.. ..........

CAE52468Ap .... ......I..S .......... ........LA .......... .I..Y..... ..M..A.K.S ..........

CAE5246Ap .... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

CAE52470Ap .... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..G....S..

ABG75614Ap .... ......I..S .......... ........LV .......... .I..Y..R.. ..M..A.K.. .......S..

CAE52485Ap .... .........S .......... ........LV .......... .I..Y..... ..M..A.K.. .......S..

FN550876Ap .... .......... .......... .......... .......... .......... .......... ..........

CAE52481Ap .... .......... .........S .........T .......... .I........ ....E..... ..........

CAE52486Ap .... .......... .......... .........T .......... .I........ .......... .L....N...

CAE52484Ap .... ..T....... .......... .........T .......... .I.T...... .S.....K.. ......N...

CAE52483Ap .... .....R.... .......... .........M .......... .I........ .G........ ......N...

ABC59575Ap .........S .......... ........LV .......G.. .I..Y..... ..M..A.K.. ..G.......

AM292923Qu .T...M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AAT80319Ap .T...M..S .......... ........LA .......... .I..Y..... ..M..A.K.. ..........

AAT80320Ap .T....M.S .......... ........LV .......... .I.AY..... ..M..A.K.. ..........

Resu

lts

163

101 193

AM498049Qu FRQVCEAFAP EARNGLVKLK YKGVFTNLFS TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKSEN EAKMSSVTTD LCI

AM494505Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM494507Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM494508Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM709777Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

FN550875Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM494511Ap .......... .......... ....S..... .......... .......... .......... .......... .......... .......... ..V

AB326230Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AB326223Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AB326227Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AB326229Ap .......... .......... .......... .......E.. .......... .......... .......... .......... .......... ...

D14996Ja .......... .......... .......... .T........ .......... .......... .......... .......... .......... ..V

P54890ApJa .......... .......... .......... .T........ .......... .......... .......... .......... .......... ..V

AM408891Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM409322Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM882704Pr .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494514Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494512Ap .......... .......... .........T ......S... .......... .......... .X........ .......... ...L...... ...

AM494509Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM709776Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494510Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AB326226Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494513Ap .......... .......... .........T ......S... .......... D......... .......... .......... ...L...... ...

AB326224Ja .......... .......... .........T ......N... .......... .......... .......... .......... ...L...... ...

ABL63752Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

ABK62735Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494506Ap .......... .......... .........T ......S... .......... .......... .......... .......... .......... ..V

AB326228Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AAT75238Pr .......... .......... .........A .......... .......... .......... .......... .......... ...L...... ...

AB326225Ap .......... ...D...... .........T .......... .......... .......... .......... .......... ...L...... ...

CAE52482Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

CAE52468Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

CAE5246ApA .......... .......... .........T ......S... .......... .......... .......... .......... ...LP..... ...

CAE52470Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

ABG75614Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

CAE52485Ap .......... .......... .........T ......S... .......... .......... .......L.. .......... ...L...... ...

FN550876Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

CAE52481Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

CAE52486Ap .......... .......... .......... .......... .F........ .......... .......... .......... .......... ...

CAE52484Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

CAE52483Ap .......... .......... .......... .......... .......... .......... ....D..... .......... .......... ...

ABC59575Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM292923Qu .......... .......... .........T ......S... ..........

AAT80319Ap .......... .......... .........T ......S... ..........

AAT80320Ap .......... .......... .........T ......S... ..........

Fig 4.16: Multiple sequence alignment of CPs (pome fruit isolates used in the study) using Multalin program (Corpet, 1988).

Resu

lts

Results

164

Fig 4.17: Radiated Phylogenetic tree drawn at amino acid level for CP (pome fruit

isolates) by Neighbor Joining method using MEGA version 4 (Tamura et al., 2007).

Indian isolates in red font.

Group 2

Group 1

Results

165

identical at nucleotide and amino acid levels respectively, and clustered in Group 1.

These isolates were obtained from trees growing in different orchards. All the other

CP (plum) isolates from the world fall in Group 2 (Fig 4.20 C, D) and shared 89-

91% sequence identity at amino acid level with the Indian plum isolates.

Phylogenetically India17 and India 18 CP isolates were relatively closer to the SX/2

plum pseudopox isolate from Poland. They shared sequence identity of 91% at the

amino acid level, well within the criteria for difference in the species (Adams et al.,

2004). Among plum isolates, the Indian sequences were unique in having valine and

phenylalanine at positions 59 and 75, respectively.

4.5.1.4 Almond

The coat protein sequence of Indian isolate of ACLSV infecting almond (isolate

India24) was unique as it had asparatic acid (D) residue at 151 position instead of

asparagine (N) when compared to other two partial CP sequences from almond (Fig

4.21 B). All the CP isolates from almond clustered in Group 2 (Fig 4.20 C, D).

4.5.1.5 Cherry

The percent sequence identity of CP sequences isolated from wild Himalayan cherry

(isolates India27, India28 and India29) with other cherry isolates at amino acid level

ranged from 86-98%. Isolates India28 and India29 amplified by IC-RT-PCR using

flower extract from the same tree shared 98% and 92-93% sequence similarity with

each other and India27 isolate respectively. The Bulgarian (P1R9D9) and Hungarian

(C-2) ACLSV-CP isolates shared maximum identity with India27 isolate. Balton-1,

the only complete CP isolate from cherry had sequence similarity of 88, 90 and 91%

with India27, India28 and India29 isolates respectively. Middle portion of the coat

protein gene shows maximum variability in amino acids (Fig 4.18 C).The India27

isolate along with the C-2, ASwC43 and P1R9D9 isolates clustered together (Fig

4.19 B) and fall in Group 1(Fig 4.20 C, D).

4.5.1.6 Apricot

CP sequences amplified from wild and cultivated apricot (isolates India26, India25)

were highly similar showing 94% identity at amino acid level. Multiple sequence

alignment of at the amino acid level showed that most of the variability was present

in the N-terminal part of the CP cistron (overlapping with the movement protein i.e.

from 60-100 amino acids) whereas the C-terminus was significantly less divergent

(Fig 4.18 D). Most of the Spanish CP (apricot) isolates clustered together while,

Results

166

India25 and India26 isolates grouped with Jordanian (AJ586631) and Italian

(AJ586630) isolates respectively (Fig 4.19 C). The India25 (cultivated apricot) falls

in Group 1 while India26 (wild apricot) belonged in Group 2 (Fig 4.20 D). Spanish

isolate Apr-109 (AJ586623) was the only isolate having Tyrosine (Y) 144 instead of

Phenylalanine (F) thus disrupting the conserved RQ/FDF residues identified for the

salt bridge structure.

4.5.1.7 Peach/Nectarine

Partial and complete CP isolates obtained from peach (Table 4.17) were compared at

amino acid level. Multiple sequence alignment indicated that maximum variability

occurred between amino acid positions 33rd and 98th (Fig 4.18 E). The India20

isolate shared 92% sequence identity with India21 and India22 isolates at amino acid

level and was the only ACLSV peach isolate falling in Group 1 with maximum

variability at amino acid level (Fig 4.18 E) and thus sharing sequence identity of less

than 87.1% with most of the peach isolates from the world. The India21 and India22

CP isolates from peach shared 98 per cent amino acid identity and clustered together

(Fig 4.19 D) in Group 2 with other peach isolates (Fig 4.20 C, D).

4.5.1.8 Pome and Stone Fruits

All the CP sequences obtained from pome and stone fruit in the present study and

those deposited earlier in GenBank (Table 4.17) were subjected to the phylogenetic

and multiple sequence analysis. The CP isolates from India shared high sequence

identity of 91-100% at aa level with world isolates. The highest degree of variability

was observed in the middle portion with 9 amino acid substitutions in contrast to the

N-terminal and C-terminal ends which were maximally conserved with only 4 amino

acid substitutions at each end. However, maximum non conserved changes creating

variability in amino acid sequences were in the middle portion of the coat protein

gene. Sequence analysis showed that India24 (almond) and India9 (apple) were the

only CP isolates having asparatic acid (D) residue at 151 position instead of

asparagine (N) (Fig 4.20 B). The isolates shared 93% sequence identity at amino

acid level and fall in Group 2 (Fig 4.20 C, D). The CP isolates India1, India3, India

4 (apple); India20 (peach); India23 (quince) and India17, 18 (plum) were the only

sequences having valine and phenylalanine amino acids at positions 59 and 75

respectively.

167

1 100


AM931534In .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

CAB46654Ge .......... .........G .......... .V....I..S .......... .......GLT .......... .I..Y..R.. .....A.KI. ...Q..N...

AAA42589Fr .......... ...S.....G ..N....... ..T...I..S .......... ........LV .........S .L..Y..... .....A.K.. ......NK..

NP040553Fr .......... ...S.....G ..N....... ..T...I..S .......... ........LV .........S .L..Y..... .....A.K.. ......NK..

AAF67188Po .......... .........V .......... .V....I..S .......... ........LM .........Q .M..Y....I ..M..A.... ......N...

101 193


AM931534In .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

CAB46654Ge .......... .......... .........T ......S... .......... .......... ........P. .......... ...L...S.. ...

AAA42589Fr .......... .......... .........T ......S... .......... .......... .......... .......... ...L...S.. ...

NP040553Fr .......... .......... .........T ......S... .......... .......... .......... .......... ...L...S.. ...

AAF67188Po .......... .......... .........T ......S... .......... .......... .......... .......... ...L...S.. ...

(A)

1 100

AE52472Ita HGKT GAILEQILES IFANIAIQGT SEQTEFLNLV VEVKSMEDQK VIGSYNLKEV VNMIKAFKTT SSDPNISSMT

CAE52465It .... .......... .......R.. .......D.M .......... ........G. ..L....... ..........

AM498046In MAAVLNLQLK VDADLKAFLA AEGRPL.... .......... .......... .......D.. .......... .......... .......... .......N..

101 193

AE52472Ita FRQICEAFAP EARNGLVKLK YNGVFTNLFA TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFARSEN EAKISSVSTD LCI

CAE52465It .......... .......... .........T .......... .......... .......... .......... .......... .......... ...

AM498046In ...V...... .......... .K.......T ......S... .......... D......... .......... ......K... ...L...T.. ...

(B)

1 100

X99752Fra_ MAAVLNLQLK VDADLKAFLA KENRPLHGKT GATLEQILES IFANIAVQGT SEHTEFLDLT VEVKSMEDQS TLGSYNLREV VNLIKAFKTT SSDPNISGMT

AM498044In .......... .......... A.G....... ..I...T..A ......I... ..Q.....V. .........K VI..F..K.. .G...I.... ......NN..

FN666579In .......... .......... A.G....... ..I....... ......M... ..Q..S...V .........K VI.....K.. ..M....... .......N..

FN666578In .......... .......... A.G....... ..I....... ......I... ..Q......V .........K VI.....K.. ..M....... .......N..

DQ329160Bu ......T..A ......I... ..Q.....V. .........K VI..F..K.. .S...I.... ......NN..

AY677106Hu .....T..A ......I... ..Q.....V. .........K VI..F..K.. .....I.... ......NN..

AY677105Hu ......... .......... .......... .......... .......G.. .......... .......R..

AY730560Tu .P....... ......I... ..Q......M .........K VI.....K.T ..M....... ...L..NN..

Contd…

Resu

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168

101 193

X99752Fra_ FRQVCEAFAP EARNGLVKLK YKGVFTNLFT TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKNES EAKISSVSTD LCI

AM498044In .......... .......... .........S .......... .......... .......... .......... .......S.N ...M...T.. ...

FN666579In .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...T.. ...

FN666578In .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...T.. ...

DQ329160Bu .......... .......... .........S .......... .......... .......... .......... .......S.N ...M...T.. ...

AY677106H .......... .......... .........S .......... ..........

AY677105Hu .......... .......... .......... .......... ..........

AY730560Tu ...L...... .......... .......... ......S... ..........

(C) 1 100

AJ586623Sp HGKT GATLELILES ILANIAIQGT SEQTEFLDLV VEVKSMEDQT VLGSYNLKEV VNLIKAFKTT SSDPNINKMT

AJ586629Sp .... ........D. .F........ .......... .......... .......... .......... .......N..

AJ586632Sp .... .......... .F........ .......... .......... .......... .......... .......N..

AJ586633Sp .... .......... .F........ .......... .......... .......... .......... .......N..

AJ586622Sp .... .......... .F........ .......... .......... I......... .......... ......SN..

AJ586634Sp .... .......... .F........ .......... .......... .......... .......E.. .......N.R

AJ586630It .... ..I..QM.K. .F........ .........A .........K .I........ ..M....... ......SS..

AJ586635It .... ..I..Q.... .F........ .........T .......... .I.....R.. ..M....M.. .......N..

AM498045In MAAVLNLQLK VDADLKAFLA AEGRPL.... ..I..QT..A .F........ ........VT .........K .I..F..... .G...I.... .......N..

AM498048In MAAVLNLQLK VDADLKAFLA AEGRPL.... ..I..Q.... .F........ .......... .........K .I........ ..M....... ......SS..

AJ586631Jo .... ..I..Q.... .F........ .........M .........K EI.......I ..M.R..R.. .......N..

101 193

AJ586623Sp FRQVCEAFAP EARNGLVKLK YKGVFTNLFT TMPEVGSKYP ELMYDFNKGL NMFIMNKTQQ KVITNMNRRL LQTEFAKSEN EAKLSSVSTD LCI

AJ586629Sp L......... .......... .......... .......... ...F...... .......A.. E......... .......... .......... ...

AJ586632Sp .......... .......... .......... .......... ...F...... .......A.. .......... .......... .......... ...

AJ586633Sp .......... .......... .......... .......... ...F...... .......A.. .......... .......... .......... ...

AJ586622Sp .......... .......... .......... .......... ...F...... .......A.. .......... .......... .......... ...

AJ586634Sp .......... .......... .......... .......... ...F...... ........R. .......... .......... .......... ...

AJ586630It .......... .......... .......... .......... ...F...... .......A.. .......... .......... .......... ...

AJ586635It .......... .......... .......... .......... ...F...... .......A.. .......... .......... .......... ...

AM498045In .......... .......... .........S ......G... ...F...... .......A.. .......... .......... ...M...T.. ...

AM498048In .......... .......... .......... .......... ...F...... .......A.. .......... .......... .......T.. ...

AJ586631Jo .......... .......... .Q........ S.....G... ...F...... .......A.. .......... .......... G......... ...

(D)

Resu

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169

1 100

AJ586646Hu HGKT GATLEQILES IFANIAVRGT SEHTEFLDLT VEVKSMEDQS TLGSYNLREV VNLIKAFKTT SSDPNISGMT

AJ586652It .... .......... ......IQ.. .........V .........K VI.....K.. ..M....... ......NN..

AJ586649Le .... ..I....... ......IQ.. ..Q......M .........R VI.....K.I ..M....... ......NN..

AJ586647Le .... ..I....... ......IQ.. ..Q......A .........K VI.....K.I ..M....... ......NN..

AJ586644It .... ..I....... ......IQ.. ..Q......M .......E.K VI.....K.T ..M....... ......ND..

AJ586648Le .... ..I...M.K. ......IQ.. ..Q......A .........K VI.....K.. ..M....... .......S..

DQ329161Bu ..I....... ......IQ.. ..Q......V .........K VI.....K.. ..M....... .......N..

AY728180Ch MAAVLNLQLK VDVDLKAFLV AEGRPL.... ..I...M... ......IQ.. ..Q......M .........K VI.....K.T ..M....R.. .......N..

AM498050In MAAVLNLQLK VDADLKAFLA AEGRPL.... ..I...M... ......IQ.. ..Q......V .........K VI.....K.. ..M....... .......N..

AM931533In MAAVLNLQLK VDADLKAFLA AEGRPL.... ..I...M... ......IQ.. ..Q......A .........K VI.....K.. ..M....... .......S..

AM498047In MAAVLNLQLK VDADLKAFLA AEGRPL.... ..I...T..A ......IQ.. ..Q.....VL .........K VV..F..K.. .....I.R.. ...T...S..

EU223295US MAATLNLQLK VDRELRAFLA EANRPL.... .G.V.L.... .......Q.. ..Q.....VE ....KSG.PT V.QK....T. .E...L.R.. ...K..NTL.

DQ329159Bu ..I....... ......IQ.. ..Q.....RV .........R VV........ .....V.... ......NN..

AY730558Tu ......... ......IQ.. ..Q......V .......... V......K.E .......... ......NN..

AY730559Tu .P....... ......IQ.. ..Q......M .........K VI.....K.T ..M....... ......NN..

AY677107Hu ......... .......Q.. .......... .......... ...C...... .......... ..........

101 193

AJ586646Hu FRQVCEAFAP EARNGLVKLK YKGVFTNLFT TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKNES EAKISSVSTD LCI

AJ586652It .......... .......... .......... ......S... .......... .......... .......... .......S.D ...V...... ...

AJ586649Le .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...... ...

AJ586647Le .......... .......... .........A .......... .......... .......... .......... .......S.N ...L...... ...

AJ586644It ...L...... .......... .......... ......S... .......... .......... .......... .......S.N ...L...... ...

AJ586648Le .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...... ...

DQ329161Bu .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...T.. ...

AY728180Ch .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...... ...

AM498050In .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...T.. ...

AM931533In .......... .......... .......... ......S... .......... .......... .......... .......S.N ...L...T.. ...

AM498047In .......... .......... .........S .......... .......... .......... .......... .......S.N ...M...T.. ..V

EU223295US ...I...... ...D...... TI......YK ......N... .......... .PML...T.R V.V..L.... .......S.N ....A...N. ...

DQ329159Bu ...I...... .......... .R......Y. .......... ......H... ..LL...... .......... .......S.N .......... ...

AY730558Tu .......... .......... .......... ......S... ..........

AY730559Tu ...L...... .......... .......... ......S... ..........

AY677107Hu .......... .......... .......... .......... ..........

(E)

Fig 4.18: Multiple sequence alignment of CP gene sequences from (A) plum, (B) almond, (C) cherry, (D) apricot and (E) peach isolates

using Multalin program (Corpet, 1988). Clustering in two groups viz. Group 1 (unshaded) and Group 2 (grey shaded). Indian isolates are in red

font, the amino acid changes are in yellow and conserved residues RQ/FDF identifying the salt bridge structure highlighted in green. The

alphabets at the end of accession numbers are initials of the country (Table 4.17).

Resu

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Results

170

(A)

(B)

(C)

(D)

Fig 4.19: Phylogenetic relationship on the basis of coat protein (CP) gene (at

amino acid level) between Indian isolates of ACLSV infecting (A) plum, (B) cherry,

(C) apricot and (D) Peach with the respective isolates of CP (listed in Table 4.17) by

Neighbor Joining method using MEGA version 4 (Tamura et al., 2007). World

isolates labeled as- accession number, country name (isolate name). Bootstrap values

(% replication) are shown on the major branches.

Results

171

Most of the CP isolates from stone fruit clustered in Group 2 suggesting co-evolution

however, isolates P1R9D9 (DQ329160, cherry) and C-2 (AY677106, cherry) were

exceptions. The Indian CP (stone fruit) isolates however, did not show such co-

evolution. Indian isolates (India17, 18) infecting plum (100% identical at amino acid

level) clustered in Group 1. These isolates were obtained from trees growing in

different orchards. Similarly, India21 (peach) and India19 (pear) isolates (sharing

100% identity) were obtained from different corners of the same orchard.

The India24 (almond) and India3 (apple) CP isolates (93% identity at amino acid

level) were from the same orchard and in close proximity to each other. However,

there were differences in phylogeny (India3- Group 1, India 24 - Group 2). The CP

isolates from plum (India17, 18; Palampur), quince (India23; Salooni), peach

(India22; Solan) and various CP isolates from apple (different locations in HP)

clustered together (Fig 4.20 D) indicating no host or region wise clustering or

similarity (Fig 4.20 D). The differences in phylogeny (clustering into Group 1 and

Group 2) were evident from pattern of variations and covariations in deduced amino

acid sequences of CP sequences (Table 4.18) from the various hosts individually (Fig

4.18, 4.19).

The India20 isolate falling in Group 1 shared sequence identity of less than 87.1%

with most of the peach isolates from the world and could be considered as the most

divergent isolate obtained in the study. The conserved residues RQ/FDF (102-

103/154-156) identified as a salt bridge structure (highlighted in green) were also

present in all the CP sequences (Fig 4.20 A, B).

4.5.1.9 Recombination Analysis: Understanding Evolutionary Relationship Based

on Coat Protein Gene

Recombination means exchange of genetic material between two genomes thus

leading to new strain. Recombination data is useful for defining evolutionary

relationships of the isolates concerned. Recombination analysis of all available

complete CP sequences from India and elsewhere gave five potential recombination

events (PREs). However, only event 1 seemed significant (Table 4.19) as it was

detected by five of the recombination detection programs viz. MAXCHI (average P-

value =3.399 x10-04), LARD (average P-value =1.166 x10-07), CHIMAERA

(average P-value =1.823x10-02), 3SEQ (average P-value = 4.481x10-06) and

BOOTSCAN (average P-value = 2.877x10-03) with identical breakpoints between

Results

172

nucleotides from 186-498 in India7 (major parent) and India12 (minor parent)

isolates. The presence of this recombination event has been depicted with the help of

a graphical representation (Fig 4.21) which clearly shows break points and location

of recombination sites. The break points were also confirmed by drawing

phylogenetic tree of 1-185,186-498 and 499-582 nt of all the Indian CP isolates. All

the sequences involved including the daughter isolate (India20, peach) fall in Group

1. In event 3 recombination in plums between French (AAA42589, major parent) and

India15 (AM882705, minor parent) isolates gave India9 (apple), India24 (apricot)

and Brazilian (apple) isolates of CP with different breakpoints as recombinant

daughters. However, this and other recombination events were detected by only one

of the recombination program and with insignificant average P-value (Table 4.19).

Recombination analysis for all the CP isolates from pome (apple, pear, quince) and

stone (almond, apricot, cherry, plum, peach) fruits was also done separately. India 1

and India18 (plum) isolates indicated towards possible minor parenting for P-863 and

PBM1 from plum isolates. All the CP sequences from peach analyzed by

Recombination Detection Program (RDP) ver. 3.26 (Martin et al., 2005) indicated

one recombination event in SISCAN programme, between the isolate HBP

(AAU93348) and India20 though with very low probability. Recombination events

with insignificant average P- values were detected by single RDP program for most

of the Indian apple isolates among themselves and with Japanese (AB326230, B6)

apple isolate. Recombination analysis performed for the CP isolates from cherry,

almond and apricot individually indicated no possible recombination event.

4.5.2 Sequence, Phylogenetic and Recombination Analysis of ACLSV

Movement Protein (MP) gene

Multiple sequence alignment of complete MP (~1405bp, 460aa) isolates (Fig 4.22A)

from India and world (Table 4.20) revealed high divergence level that was unevenly

distributed all along the 50K protein. Complete MP sequences showed some

conservation in N-terminal portion (230aa) except few changes, while C-terminal

half showed a much higher variability, with only a few blocks of fully conserved

residues. Percent similarities among deduced amino acid sequences of MP gene of

various ACLSV isolates are shown in Table 4.21. The Indian MP (FN673831,

India15Apmp) isolate showed 61-86% similarity at amino acid level with other

isolates from the world. The Indian isolate was found to be maximally (86%) similar

173

1 100

FN666579Ch MAAVLNLQLK VDADLKAFLA AEGRPLHGKT GAILEQILES IFANIAMQGT SEQTESLDLV VEVKSMEDQK VIGSYNLKEV VNMIKAFKTT SSDPNISNMT

FN666578Ch .......... .......... .......... .......... ......I... .....F.... .......... .......... .......... ..........

AM498047Pe .......... .......... .......... ......M... ......I... .....F.... .......... .......... .......... ..........

AM498048Ar .......... .......... .......... .......... ......I... .....F.... .......... .......... .......... .......S..

AM498046Al .......... .......... .......... .......... ......I... .....F.... .......... .......... .......... ..........

AM931533Pe .......... .......... .......... ......M... ......I... .....F...A .......... .......... .......... .......S..

AAF67188Pl .......... .........V .......... .V........ ......I... .....F...M .........Q .M.......I .......R.. ......N...

AY728180Pe .......... ..V......V .......... ......M... ......I... .....F...M .......... .........T .......R.. ..........

AAA42589Pl .......... ...S.....G ..N....... ..T....... ......I... .....F.... .........S .L........ ..L....... ......NK..

NP_040553Pl .......... ...S.....G ..N....... ..T....... ......I... .....F.... .........S .L........ ..L....... ......NK..

AM882705Pl .......... .......... .......... ......T..A ......I... .....F..VL .......... .V..F..... ..L..I.R.. ..........

AM931534Pl .......... .......... .......... ......T..A ......I... .....F..VL .......... .V..F..... ..L..I.R.. ..........

AM498050Pe .......... .......... .......... ......T..A ......I... .....F..VL .......... .V..F..... ..L..I.R.. ...T...S..

AM498044Ch .......... .......... .......... ......T..A ......I... .....F..VT .......... ....F..... .GL..I.... ......N...

AM498045Ar .......... .......... .......... ......T..A ......I... .....F..VT .......... ....F..... .GL..I.... ......N...

CAB46654Pl .......... .........G .......... .V........ ......I... .....F.G.T .......... .......R.. ..L.....I. ...Q..N...

X99752Che .......... .......... K.N....... ..T....... ......V... ..H..F...T .........S TL.....R.. ..L....... .......G..

EU223295Pe ...T...... ..RE.R.... EAN....... .GTV.L.... ......V... .....F..VE ....KSG.PT .LQK...RT. .EL..L.R.. ...K..NTL.

AJ586652Pe .... ..T....... ......I... ..H..F.... .......... .......... .......... ......N...

AJ586649Pe .... .......... ......I... .....F...M .........R .........I .......... ......N...

AJ586647Pe .... .......... ......I... .....F...A .......... .........I .......... ......N...

AJ586635Ar .... .......... ......I... .....F...T .........T .......R.. .......M.. ......N...

AJ586644Pe .... .......... ......I... .....F...M .......E.. .........T .......... ......ND..

AJ586648Pe .... ......M.K. ......I... .....F...A .......... .......... .......... .......S..

AJ586630Ar .... ......M.K. ......I... .....F...A .......... .......... .......... .......S..

AJ586623Ar .... ..T..L.... .L....I... .....F.... .........T .L........ ..L....... ......NK..

AJ586629Ar .... ..T..L..D. ......I... .....F.... .........T .L........ ..L....... ......N...

AJ586632Ar .... ..T..L.... ......I... .....F.... .........T .L........ ..L....... ......N...

AJ586633Ar .... ..T..L.... ......I... .....F.... .........T .L........ ..L....... ......N...

AJ586622Ar .... ..T..L.... ......I... .....F.... .........T IL........ ..L....... ..........

AJ586634Ar .... ..T..L.... ......I... .....F.... .........T .L........ ..L....E.. ......N..R

AE52472Al .... .......... ......I... .....F.N.. .......... .......... .......... .......S..

CAE52465Al .... .......... ......IR.. .....F...M .......... ........G. ..L....... .......S..

AJ586631Ar .... .......... ......I... .....F...M .......... E........I ....R..R.. ......N...

AJ586646Pe .... ..T....... ......VR.. ..H..F...T .........S TL.....R.. ..L....... .......G..

DQ329161Pe .......... ......I... .....F.... .......... .......... .......... ..........

DQ329160Ch ..T...T..A ......I... .....F..VT .......... ....F..... .SL..I.... ......N...

DQ329159Pe .......... ......I... .....F..R. .........R .V.....R.. ..L..V.... ......N...

AY730558Pe .T....... ......I... .....F.... .........S .L.......E ..L....... ......N...

AY730559Pe .P....... ......I... .....F...M .......... .........T .......... ......N...

AY730560Ch .P....... ......I... .....F...M .......... .........T .......... ...L..N...

AY677106Ch .T...T..A ......I... .....F..VT .......... ....F..... ..L..I.... ......N...

AY677107Pe .T....... ......V... ..H..F...T .........S TL.C...R.. ..L....... .......G..

AY677105Ch .T....... ......V... ..H..F...T .........S TL.....G.. ..L....... .......R..

Resu

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101 193

FN666579Ch FRQVCEAFAP EARNGLVKLK YKGVFTNLFT TMPEVGSKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKSEN EAKLSSVTTD LCI

FN666578Ch .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM498047Pe .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM498048Ar .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM498046Al .......... .......... .......... .......... .......... D......... .......... .......... .......... ...

AM931533Pe .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AAF67188Pl .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AY728180Pe .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AAA42589Pl .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

NP_040553Pl .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AM882705Pl .......... .......... .........S ......G... .......... .......... .......... .......... ...M...... ...

AM931534Pl .......... .......... .........S ......G... .......... .......... .......... .......... ...M...... ...

AM498050Pe .......... .......... .........S ......G... .......... .......... .......... .......... ...M...... ..V

AM498044Ch .......... .......... .........S ......G... .......... .......... .......... .......... ...M...... ...

AM498045Ar .......... .......... .........S ......G... .......... .......... .......... .......... ...M...... ...

CAB46654Pl .......... .......... .......... .......... .......... .......... ........P. .......... .......S.. ...

X99752Che .......... .......... .......... ......G... .......... .......... .......... .......N.S ...I...S.. ...

EU223295Pe ...I...... ...D...... TI......YK ......N... .......... .PML...T.R V.V..L.... .......... ...IA..SN. ...

AJ586652Pe .......... .......... .......... .......... .......... .......... .......... .........D ...V...S.. ...

AJ586649Pe .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586647Pe .......... .......... .........A ......G... .......... .......... .......... .......... .......S.. ...

AJ586635Ar .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586644Pe ...L...... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586648Pe .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586630Ar .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586623Ar .......... .......... .......... .......... ...Y...... .......T.. .......... .......... .......S.. ...

AJ586629Ar L......... .......... .......... .......... .......... .......... E......... .......... .......S.. ...

AJ586632Ar .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586633Ar .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586622Ar .......... .......... .......... .......... .......... .......... .......... .......... .......S.. ...

AJ586634Ar .......... .......... .......... .......... .......... .......TR. .......... .......... .......S.. ...

AE52472Al ...I...... .......... .N.......A ......G... .......... .......... .......... ......R... ...I...S.. ...

CAE52465Al ...I...... .......... .N........ ......G... .......... .......... .......... ......R... ...I...S.. ...

AJ586631Ar .......... .......... .Q........ S.....G... .......... .......... .......... .......... G......S.. ...

AJ586646Pe .......... .......... .......... ......G... .......... .......... .......... .......N.S ...I...S.. ...

DQ329161Pe .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

DQ329160Ch .......... .......... .........S ......G... .......... .......... .......... .......... ...M...... ...

DQ329159Pe ...I...... .......... .R......Y. ......G... ......H... ..LL...... .......... .......... ...I...S.. ...

AY730558Pe .......... .......... .......... .......... ..........

AY730559Pe ...L...... .......... .......... .......... ..........

AY730560Ch ...L...... .......... .......... .......... ..........

AY677106Ch .......... .......... .........S ......G... ..........

AY677107Pe .......... .......... .......... ......G... ..........

AY677105Ch .......... .......... .......... ......G... ..........

(A)

Resu

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1 100 AM498049Qu MAAVLNLQLK VDADLKAFLA AEGRPLHGKT GAILEQTLEA IFANIAIQGT SEQTEFLDVL VEVKSMEDQK VVGSFNLKEV VNLIKIFRTT SSDPNISNMT

AM494505Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494507Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494508Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM882705Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM931534Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM709777Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

FN550875Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AM494511Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AB326230Ap .......... .......... .......... .......... .......... .........Q .......... .M........ .......... .......K..

AB326223Ap .......... .......... .......... .......... .......... .........M .......... .I........ .S.....K.. ......N...

AB326227Ap .......... ...E...... .......... .......... .......... .........T .......... .I........ .S.V...K.. ......N...

AB326229Ap .......... .......... .......... .......... .......... .........T .......... .I........ .......K.. ......N...

AM498044Ch .......... .......... .......... .......... .......... .........T .......... .I........ .G.....K.. ......N...

AM498045Ar .......... .......... .......... .......... .......... .........T .......... .I........ .G.....K.. ......N...

D14996Ap .......... .......... .......... .......... .......... .......... .......... .......... .G........ .......S..

P54890Ap .......... .......... .......... .......... .......... .......... .......... .......... .G........ .......S..

AM408891Ap .......... .......... .......... .......... .......... .........T .......... .......... .......... ...T...S..

AM498047Pe .......... .......... .......... .......... .......... .......... .......... .......... .......... ...T...S..

AM409322Ap .......... .......... ....S..... .......... .......... .........T .......... .......... .......... ...T...S..

FN666579Ch .......... .......... .......... ......I..S ......M... .....S..LV .......... .I..Y..... ..M..A.K.. ..........

FN666578Ch .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494510Ap .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494509Ap .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM709776Ap .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM498050Pe .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM494513Ap .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AM498046Al .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AB326224Ap .......... .......... .......... ......I..S .......... ........LM .......... .I..Y..... ..M..A.K.. ..........

ABL63752Ap .......... .......... ........R. ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. .......S..

AM498048Ar .......... .......... .......... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. .......S..

AM882704Pr .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM494514Ap .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM931533Pe .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AM494512Ap .......... .......... .......... ......M..S .......... ........LA .......... .I..Y..... ..M..A.K.. .......S..

AB326226Ap .......... .......... .......... ......M..S .......... ........LS .......... .I..Y..... ..M..A.K.. ..........

ABK62735Ap .......... .......... .......... .........S .......... ........LV .........E .I..Y..... ..M..A.K.. ..........

AM494506Ap .......... .......... .......... ......M..S .......... ........LV .......... .I..Y..... ..M..A.K.. ...S......

AAF67188Pl .......... .........V .......... .V....I..S .......... ........LM .........Q .M..Y....I ..M..A.... ......N...

AY728180Pe .......... ..V......V .......... ......M..S .......... ........LM .......... .I..Y....T ..M..A.... ..........

AB326228Ap .......... .......... .D-....... ......I..S .......... ........LV .......... .I..Y..... ..M..A.K.. ..........

AAT75238Pr .......... .....R.... ...T...... .........S ....V..... ........MM ......D... .IS.Y...S. .D...V.... ......NG..

AB326225Ap .......... ......V... .......... .....L...S .......... ........LV .......... ..N.Y...S. .D.....K.. ......NG..

AAA42589Pl .......... ...S.....G ..N....... ..T...I..S .......... ........LV .........S .L..Y..... .....A.K.. ......NK..

NP_040553Pl .......... ...S.....G ..N....... ..T...I..S .......... ........LV .........S .L..Y..... .....A.K.. ......NK..

X99752Ch .......... .......... K.N....... ..T...I..S ......V... ..H.....LT .........S TL..Y..R.. .....A.K.. .......G..

CAB46654Pl .......... .........G .......... .V....I..S .......... .......GLT .......... .I..Y..R.. .....A.KI. ...Q..N...

EU223295Pe ...T...... ..RE.R.... EAN....... .GTV.LI..S ......V... .........E ....KSG.PT .LQKY..RT. .E...L.... ...K..NTL.

FN550876Ap .... .......... .......... .......... .......... .......... .......... ..........

Resu

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101 193 AM498049Qu FRQVCEAFAP EARNGLVKLK YKGVFTNLFS TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKSEN EAKMSSVTTD LCI

AM494505Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM494507Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM494508Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM882705Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM931534Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM709777Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

FN550875Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM494511Ap .......... .......... ....S..... .......... .......... .......... .......... .......... .......... ..V

AB326230Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AB326223Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AB326227Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AB326229Ap .......... .......... .......... .......E.. .......... .......... .......... .......... .......... ...

AM498044Ch .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

AM498045Ar .......... .......... .......... .......... .......... .......... .......... .......... .......... ...

D14996Ap .......... .......... .......... .T........ .......... .......... .......... .......... .......... ..V

P54890Ap .......... .......... .......... .T........ .......... .......... .......... .......... .......... ..V

AM408891Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM498047Pe .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

AM409322Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

FN666579Ch .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

FN666578Ch .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494510Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494509Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM709776Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM498050Pe .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494513Ap .......... .......... .........T ......S... .......... D......... .......... .......... ...L...... ...

AM498046Al .......... .......... .........T ......S... .......... D......... .......... .......... ...L...... ...

AB326224Ap .......... .......... .........T ......N... .......... .......... .......... .......... ...L...... ...

ABL63752Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM498048Ar .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM882704Pr .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494514Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM931533Pe .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494512Ap .......... .......... .........T ......S... .......... .......... .X........ .......... ...L...... ...

AB326226Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

ABK62735Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AM494506Ap .......... .......... .........T ......S... .......... .......... .......... .......... .......... ..V

AAF67188Pl .......... .......... .........T ......S... .......... .......... .......... .......... ...L...S.. ...

AY728180Pe .......... .......... .........T ......S... .......... .......... .......... .......... ...L...S.. ...

AB326228Ap .......... .......... .........T ......S... .......... .......... .......... .......... ...L...... ...

AAT75238Pr .......... .......... .........A .......... .......... .......... .......... .......... ...L...... ...

AB326225Ap .......... ...D...... .........T .......... .......... .......... .......... .......... ...L...... ...

AAA42589Pl .......... .......... .........T ......S... .......... .......... .......... .......... ...L...S.. ...

NP_040553Pl .......... .......... .........T ......S... .......... .......... .......... .......... ...L...S.. ...

X99752Ch .......... .......... .........T .......... .......... .......... .......... .......N.S ...I...S.. ...

CAB46654Pl .......... .......... .........T ......S... .......... .......... ........P. .......... ...L...S.. ...

EU223295Pe ...I...... ...D...... TI......YK ......N... .......... .PML...T.R V.V..L.... .......... ...IA..SN. ...

FN550876Ap .......... .......... .......... .......... .......... .......... .......... .......... .......... ..V

(B) Resu

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177

(C)

Group 2

Group 1

Resu

lts

178

Group 2 Group 1

(D)

Fig 4.20: Multiple sequence alignment of CP gene sequences at amino acid level (A) all the stone fruit isolates (B) all the complete CP

isolates from pome and stone fruits using Multalin program (Corpet, 1988). Clustering in two groups viz. Group 1 (unshaded) and Group 2 (grey

shaded). Indian isolates are in red font, the amino acid changes are in yellow while the conserved residues RQ/FDF identifying the salt bridge

structure highlighted in green. The alphabets at the end of accession numbers are the abbreviated hosts Ap-apple, Al-almond, Ar-apricot, Ch-

cherry, Pe-peach, Pl-plum, Pr-pear, Qu-quince (Table 4.17). Phylogenetic relationship on the basis of coat protein (CP) gene sequence at amino

acid level of all the CP isolates (Indian and world) used in the study (C) Radiated phylogenetic showing two major groups (D) Rectangular

phylogenetic tree drawn by Neighbor Joining method using MEGA version 4 (Tamura et al., 2007) with 50% bootstrap cut-off value.

Resu

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Results

179

Table 4.19: Recombination analysis results for all the available complete CP

sequences used in the study. Indian isolates in italics.

Even

t No.

Major

Parent

Minor

Parent Daughter

Breakpoint

s Detected By

Average

P- Value

1. India7

(apple)

India12

(apple)

India20

(peach) 186-498

BOOTSCAN 2.877x10-03

MAXCHI 3.399 x10-04

CHIMAERA 1.823x10-02

LARD 1.166 x10-07

3SEQ 4.481x10-06

2.

P-863

M58152

(plum)

India3

(apple)

India24

(almond)

26-211 SISCAN 2.344x10-02

P-863

M58152

(plum)

India3

(apple)

India25

(apricot) 26-211 SISCAN 2.344x10-02

3.

P-863

M58152

(plum)

India15

(apple)

India9

(apple) 26-211 SISCAN 9.724x10-04

ABL63752

(apple) 104-533 SISCAN 9.724x10-04

India26

(apricot) 104-533 SISCAN 9.724x10-04

4.

B6

AB326224

(apple)

India7

(apple)

P-863

M58152

(plum)

23-104 GENECOV 4.413x10-04

B6

AB326224

(apple)

India7

(apple)

NC_001409

(plum) 23-104 GENECOV 4.413x10-04

5. India20

(peach)

India7

(apple)

P-863

M58152

(plum)

333-538 SISCAN 6.223x10-04

1 145 291 436 582

Position in alignment

0.0000

0.0260

0.0530

0.0800

0.1070

Tract of sequence with a recombinant origin

AM494511 Ap India7 - AM409322 Ap India12

AM494511 Ap India7 - AM498047 Pe India20

AM409322 Ap India12- AM498047 Pe India20

Bonferroni corrected P-Value = 0.05

Fig 4.21: Distance plot showing recombination between AM494511 (India7,

major parent, apple) and AM409322 (India12, minor parent, apple) and formation of

daughter isolate (India20, peach, AM498047) when all the complete CP isolates were

analysed by Recombination Detection Program (RDP) ver. 3.26 (Martin et al., 2005).

Results

180

to P-205 (apple; D14996) and least (61%) to TaTao-5 (peach, EU223295) isolate. As

expected the Indian MP isolate at 5’-end had two adjacent independently active

conserved RNA-binding domains (A; 82-126 and B; 127-287aa) with ‘D/G’ motifs

characteristic of ‘30K Super Family’ of movement proteins (Fig 4.22A). The amino

residues underlined in domains A and B show the principal conserved motif and a

region of positively charged residues (Fig 4.22 A).

The phylogenetic analysis showed that Indian isolate and MO-5 (apple) isolate were

more closely related though sharing only 81% sequence similarity at amino acid

level. However, B6 apple isolate having 84% identity to Indian isolate shared

phylogeny with stone fruit isolates and was the only apple isolate in stone fruit

cluster. Three clusters viz. apple, stone fruit and isolates having very different

phylogeny (Balton-1, TaTao-5) were identified (Fig 4.22 B).

To have better insight into the sequence pecularities among various MP isolates the

5’-end and 3’-end sequences of MP overapping with the replicase (~67nt) and CP

(~316nt) regions respectively were analysed separately. Analysis of first 200aa of the

5’-end of MP sequence [8 world and 3 Indian isolates; Table 4.20) at phylogenetic

level indicated that the conserved domains and D/G motiff were present unaltered in

all three Indian isolates. The isolates were maximaly conserved however, sequences

variabilty w.r.t. host (pome and stone fruit isolates was evident (Fig 4.23 A). Most of

the MP isolates (apple) from India and world clustered together. TaTao5 MP isolate

showed clear difference in phylogeny compared to the rest (Fig 4.23 B). The 5’-end

(~200aa) of Indian MP isolates FN673831 (India15Apmp) and AM888396

(India11Apmp isolate) having sequene identity of 95% clustered together (Fig 4.23

B) and shared maximum sequence similarity of 92% and 97% at amino acid level

with P-205 (apple; D14996) isolate respectively. The IndiaAp15mp and IndiaAp RC-

Plp isolate (sharing 91% identity) were unique from rest in having certain major aa

changes (Fig 4.23 A) in the replicase-MP overlapping (~67nt / 22aa) region listed in

Table 4.22. Most of the amino acid changes were conserverd except those at aa

positions 20 and 22 in the IndiaAp RC-Plp isolate (AM888397). The IndiaAp RC-

Plp isolate was phlogenetically closer to stone fruit isolates and distant from the other

Indian MP isolates.

Apart from this deduced amino acid sequence of 3’-end of MP (110aa) overlapping

with the CP were obtained by frame shift reading of 29 CP isolates from India.

Sequence analysis using ClustalW revealed that all these Indian isolates among

Results

181

themselves shared identity ranging from 57-100% at amino acid basis indicating

maximum variabilty in the C-terminal half (Fig 4.23 C). Phylogenetic relationship of

the 3’-end of MP isolate showed separation into Group 1 and Group 2 clusters

identified for CP isolates. Group 1 isolates were unique in having Glycine

(hydrophobic) instead of Valine (polar) and Arginine (R, basic)/Glutamic acidic (E,

acidic) at 395th and 438

th amino acid (aa) positions respectively (Fig 4.23 C).

However, partial ACLSV-MP (351-460 aa) isolates viz. MO-5, Balton-1, TaTao5,

India27 and India15Apmp show intermediate sequences identities to both the groups

like recorded in analysis of 5’end MP sequences. It was evident from Balton-1

isolate having Glycine as 438th

aa residue like Group 1 isolates though it clusters

seperately. Similarly rest of these isolates also cluster somewhat separately from the

two groups (Fig 4.23 D). All the Group 2 isolates show more variability in 351-377

aa as compared to Group 1 isolates (Fig 4.23 C).

Recombination analysis for complete MP sequences indicated maximum

recombination signals at 3’-end of MP which is least conserved (Fig 4.22 A) and

overlaps with CP gene (~316nt). These events had significant average P-values but

were detected by only one of the programmes and hence considered inconsequential.

Similarly the breakpoints at 839-946 and 895-966nt positions towards the 3’-end of

MP (not in the MP-CP overlapping portion) were detected by three softwares

however, with insignificant average P-values (Table 4.23). From the 5’-end two

significant events (detected by four detection programmes each) (breakpoints 6-27

and 4-18nt) were recorded. However, the 6-27nt region included only 5 overlapping

nucleotides (nt) between replicase and MP. While, 4-18nt region was not included in

the replicase-MP overlapping portion and could be considered a PRE of 3-end of

replicase. One of the significant events in overlapping region has been depicted with

the help of a graphical representation (Fig 4.24) which clearly shows break points

and location of recombination sites.

Recombination events were also detected separately by analyzing 5’- and 3’-end

isolates of MP. About 80 potential recombination events were detected on analysis of

5’-end isolates. Of these, most were insignificant (detected by less than three

programmes). However, only one event was detected by four programmes

simultaneously with breakpoints at 6th and 27

th nt positions as obtained on complete

MP sequence analysis (Table 4.23). Recombination events were also detected from

the 3’end isolates of MP overlapping CP. However, these were with less significant

Results

182

average P-values and validated by only two recombination programmes. One such

event (detected by only two softwares) from analysis of 3’end MP isolates was worth

mention as it resulted in the recombinant daughter India20 isolate by similar

recombination (India7 major and India12 minor parenting) as reported for

recombination analyses for CP isolates (Table 4.19). The breakpoints were in

different region having only 17nt common with the CP recombination study clearly

indicating MP-CP overlapping area as a recombination region.

By aligning complete MP (FN673831) and CP (FN550875) sequences of India15

(apple) isolate amplified in the study, two probable hypothetical sequences

(~1856bp, Fig 4.25) viz. India15Ap-1 and India15Ap-2 (sharing 98% sequence

identity at nucleotide level) could be obtained. The phylogenetic analysis at

nucleotide level of this complete MP-CP region showed distinct clustering of apple

and stone fruit isolates (Fig 4.26). Recombination results show more recombination

events in MP-CP overlapping region (316nt) than in replicase-MP (~67nt) region. As

obtained by earlier analysis, the recombination events at MP-CP overlapping region

were relatively significant, clearly indicating that the former region is important with

respect to recombination and subsequent evolution. Two significant events (with

break points 1753-1820 and 1732-1828) were also detected in the 3’-end non/un-

translated region (NTR /UTR) region (Table 4.23).

Fig 4.26: Phylogenetic tree drawn with Tree Explorer ver. 2.12 (Tamura, 1999)

showing relationship between complete 1836bp (containing MP-CP genes and

3’UTR) sequences from various host and countries.

Results

183

Table 4.20: All the ACLSV-MP sequences used for analysis

S. no. Accession no. Country/Region Isolate name Length Source

1. EU223295 USA TaTao5 Complete (C) peach (Pe)

2. D14996 Japan (Jap) P-205 C apple (Ap)

3. AB326225 Japan (Jap) MO-5 C apple (Ap)

4. AB326224 Japan (Jap) B6 C apple (Ap)

5. M58152 France (Fra) P863 C plum (Pl)

6. NP_040553 France (Fra) - C plum (Pl)

7. AJ243438 Germany (Ger) PBM1 C plum (Pl)

8. X99752 France (Fra) Balton-1 C sweet cherry (Che)

9. FN673831 India India15Apmp C apple (Ap)

10. AM888397 Palampur IndiaAp RC-

Plp

Partial (P)

(5’end)

apple (Ap)

11. AM888396 Kinnaur India11Apmp P (5’end) apple (Ap)

12. AM494505 Nagri India1 P (3’end) apple (Ap)

13. AM494506 Dobi India2 P (3’end) apple (Ap)

14. AM494507 Solan India3 P (3’end) apple (Ap)

15. AM494508 Nihari India4 P (3’end) apple (Ap)

16. AM494509 Kalpa India5 P (3’end) apple (Ap)

17. AM494510 Bajaura India6 P (3’end) apple (Ap)

18. AM494511 Tissa India7 P (3’end) apple (Ap)

19. AM494512 Sangla India8 P (3’end) apple (Ap)

20. AM494513 Salooni India9 P (3’end) apple (Ap)

21. AM494514 Palampur India10 P (3’end) apple (Ap)

22. AM408891 Kinnaur India11 P (3’end) apple (Ap)

23. AM409322 Kotgarh India12 P (3’end) apple (Ap)



26. FN550875 Kashmir India15 P (3’end) apple (Ap)

27. FN550876 Uttrakhand India16 P (3’end) apple (Ap)

28. AM882705 Palampur India17 P (3’end) Plum (Pl)

29. AM931534 Palampur India18 P (3’end) Plum (Pl)

30. AM882704 Palampur India19 P (3’end) Pear (Pr)

31. AM498047 Kullu India20 P (3’end) Peach (Pe)

32. AM931533 Palampur India21 P (3’end) Peach (Pe)

33. AM498050 Solan India22 P (3’end) Peach (Pe)

34. AM498049 Salooni India23 P (3’end) quince (Qu)

35. AM498046 Solan India24 P almond (Ald)





Results

184

S. no. Accession no. Country/Region Isolate name Length Source

36. AM498045 Solan India25 P apricot (Apr)

37. AM498048 Kullu India26 P wild apricot (Apr)

38. AM498044 Palampur India27 P Wild Himalayan Cherry (Che)

39. FN666579 Palampur India28 P -do-

40. FN666578 Palampur India29 P -do-

41. AAF86667

(CMLV) Canada

C Prunus avium

Table 4.21: Percentage sequence identities of complete MP gene at amino acid

level (below diagonal) among different ACLSV isolates using ClustalW.

FN

67

38

31

Ind

ia1

5A

pm

p

M5

81

52

(P

86

3)

Pl

NC

_0

01

40

9 P

l

AB

32

62

25 (

Mo

-5)

Ap

EU

22

32

95 (

Ta

Tao 5

) P

e

AB

32

62

24 (

B6

) A

p

AJ

24

438

(P

BM

1)

Pl

X9

97

52

(B

alt

on

-1)

Ch

e

D1

49

96

(P

-20

5)

Ap

FN673831 India15Apmp X

M58152 (P863) Pl 80 X

NC_001409 Pl 80 100 X

AB326225 (MO-5) Ap 81 79 79 X

EU223295 (TaTao 5) Pe 61 60 60 60 X

AB326224 (B6) Ap 84 84 84 82 61 X

AJ24 438 (PBM1) Pl 83 84 84 80 60 86 X

X99752 (Balton-1) Che 77 77 77 78 60 78 80 X

D14996 (P-205) Ap 86 79 79 80 61 83 83 79 X

Results

185

Table 4.22: Unique amino acids changes in Indian MP isolates at the 5’-end

overlapping with the Replicase gene. Changes in red font are semi-conserved while

rests are conserved changes. (R-Arginine, H-Histidine, Q-Glutamine, A-Alanine, V-

Valine, S-Serine, E-Glutamic acid, N-Arginine, K-Lysine, G-Glycine).

S. no. Accession

no.

Amino acid position

6 8 11 20 22 29 43 45

1. FN673831 R H to R R to Q A V S to E H to Q N

2. AM888397 R to K H R A to V V to G S H N to K

Fig 4.25: Graphical representation of combined MP-CP region of 1856bp

obtained from Indian amplicons. Primers used for amplification are in green font.

Points of initiation are indicated by block arrows and position in red font. MP-

movement protein, CP-coat protein, up-forward primer, dn-reverse primer.

Results

186

Table 4.23: Recombination analysis results for all the MP sequences analyzed in the

study. Indian isolates are in italics.

Location

analyzed Major Parent

Minor

Parent Daughter

Break

points Detected By

Average

P- Value

5’end

(1-443nt)

FN673831

India15Apmp

apple

EU223295

(TaTao-5)

Peach

AB326225

(MO-5)

apple

6-27

RDP 1.458 x10-05

GENECONV 2.804 x10-03

MAXCHI 2.121 x10-02

CHIMAERA 1.131 x10-02

FN673831

India15Apmp

apple

AB326225

(MO-5)

apple

AM888397

IndiaAp

RC-Plp

apple

45-160

BOOTSCAN 4.526 x10-01

MAXCHI 2.376 x10-02


Complete

MP

(~1408bp)

FN673831

India15Apmp

apple

EU223295

(TaTao-5)

Peach

AB326225

(MO-5)

apple

6-27

RDP 1.566x10-06


MAXCHI 2.522 x10-02


FN673831

India15Apmp apple

EU223295

(TaTao-5) Peach

AB326224 (B6) apple

839-946

RDP 3.178 x10-02



NC_001409

plum

FN673831

India15Apmp

apple

X99752

(Balton-1)

cherry

895-966

RDP 1.824 x10-02


CHIMAERA 7.271x10-03

M58152

(P863) Plum

FN673831

India15Apmp

apple

AB326225

(MO-5)

apple

4-18

RDP 1.566x10-06


MAXCHI 2.522 x10-02


3’end

(1073-

1408nt)

~333bp

India7

(apple)

India12

(apple)

India20

(peach) 19-202

MAXCHI 7.070 x10-03

3SEQ 4.518x10-02

MP-CP

(1-1856)

X99752

(Balton-1)

cherry

FN673831

India15Ap-2

AB326225

(MO-5)

apple

1753-

1820

RDP 1.040x10-03



SISCAN 3.114x10-02

AB326225

(MO-5)

apple

FN673831

India15Ap-1

NC_001409

plum

1732-

1828

RDP 1.259x10-02

GENECONV 1.596x10-02


MAXCHI 7.118 x10-09


FN673831 India15Ap-1

FN673831 India15Ap-2

AB326225 (MO-5)

apple

1097-1400

GENECONV 1.582x10-02

SISCAN 5.077x10-09

187

1 * 100

AB326225Ap MMIRGHKL KIAEGDIPIA GVKSSRIYSD VSPFKRASDL MIHWNEFVFK VMPEDIAGDG FRLASIPVIP DSEVKAVIRK RDSTNYVHWG ALSISIDALF

NC_001409Pl MAT.....R. R......... .......... I....K.... .......... .......... .......... S...Q..L.. .E........ ..........

M58152Pl MAT.....R. R......... .......... I....K.... .......... .......... .......... S...Q..L.. .E........ ..........

AB326224Ap ........ R......... .......... I......... .......... ...D...... .......... S...Q..L.. .E........ ..........

AJ243438Pl MAT....... .......... .......... .......... .......... .......... .....M.... S...Q..L.. .E........ ..........

D14996Ap ........ .......... .......... ....RK.... .......... .......... .......... S...Q..L.. .E........ ..........

FN673831In MA.....R.. Q..V...... ........E. .......... ..Q....... .......... .......... S...Q..L.. .EG....... ..........

X99752Che MAL....... .......... .......... I......... .......... .......... .......... ST..Q..L.. .E........ ..........

EU223295Pe MA..V....S .......... ..R....... IT........ .......... ......V.K. .....V..V. I..IQS.L.. .EG....... ..........

Consensus mam.!..hkl k..e...... ..k.....s. !s..kr.... ..h....... ...#..a.d. .....i..!. ss.!qa.l.. .#s....... ..........

101 * * * * 200

AB326225Ap KKNAGVSGWC YVYDNRWETF EQAKLQKFRF NLDSGSATLV TSPNFPVSLD DPGLSNSICV AVMFENLNFK LDSYPISVRV GTMCRFFDSF LSNVKNKNDS

NC_001409Pl R......... .......... ...M....H. .......... .......... ........S. .......... FE........ .N........ ..S....V..

M58152Pl R......... .......... ...M....H. .......... .......... ........S. .......... FE........ .N........ ..S....V..

AB326224Ap R......... .......... ...I...... ........F. .......... ........S. .......... .E........ .N...I...L ..S.R..VE.

AJ243438Pl R......... .......... ...M....H. .......... .......... ........S. .......... .E........ .N........ ..S....V..

D14996Ap R......... .......... ...M...... .......... .......... ........S. .......... .E........ .N........ ..C....V..

FN673831In R......... .......... ...M...... .......... .......... ........S. .......... .E........ .N........ ..S....V..

X99752Che R......... .......... ...M....H. ........M. .......... ........S. .......... .E........ .N........ ..S...RV..

EU223295Pe ......T.Q. ..F.K..T.. D..L....E. ...R.....I ....SS.... ....MD.... .......... .EN....... .N........ ..S.R..DE.

Consensus r.....s.w. ..%.n..e.. #..m...... ...s....l! ....fp.... ....s#..s. .......... l#s....... .n...f...f ..s.k.kv#.

201 300

AB326225Ap NFLLEASNAD PLGASVFGFD DDDQVSELFN YIQTVPTQAI KSREHEIPRG MFGIMGKKTV KSFEFASGSK GLNKIKPQRG RSSGRSSSQR FAPGFKSQDE

NC_001409Pl .......... ...VGA...E Q......... .......... .F......K. FL.M....KI .......... .MERR..N.. KQID..F... AV...R..N.

M58152Pl .......... ...VGA...E Q......... .......... .F......K. FL.M....KI .......... .MERR..N.. KQID..F... AV...R..N.

AB326224Ap ......A..E ....GA...E Q......... .......... ........K. L.......K. .T.......R SMERRR.... .PLE..T... VV........

AJ243438Pl ......A... ...VGA...E Q......... ..H....... ........K. L..ML...K. .........R SMGR...Y.. MQFE...... IV...R...D

D14996Ap ......A... ....GA...E Q......... .......... ......V... L..M....K. .........R N.GRR..... .PLE..A.L. V....R....

FN673831In ......A... ....GA...E Q......... .......... .......... L..M....K. .......... SMERRR.... .PLE...... VI........

X99752Che .....T.... ....GA...E N........D .......... ....L...K. F..ML...K. .......E.R NK..T..L.. .PAT..A... EL......AD

EU223295Pe .ARI..A..E ...LAD...E GG.RI....D .V.S..VM.V QTK.M...K. L..L...R.. .....T.KAG NARRREWSKL KI..DVAGVK -L.A.GGEF.

Consensus .fll.aa..# ...aga...# qd.q!....# .!qt..tq.! ksr.h.!.k. lf.m$..kk! .s...a.gs. ...rrkp.rg r...rs.sqr ...g..s#d#

301 400

AB326225Ap KVEHQGLATD LDFENFLKDE GRRKAGSKSI ASEGSSVDNI SSREFQFARQ NQAKENGGSS KSSTKGGRRS ESVPGRRRQT PSWKDRGNSG TDTGVHLREH

NC_001409Pl .......S.. S......RNK RGN...V..T .......... .......... .....D.S.. EFAAQ...K. KGIS...K.. S.......P. ..........

M58152Pl .......S.. S......RNK RGN...V..T .......... .......... .....D.S.. EFAAQ...K. KGIS...K.. S.......P. ..........

AB326224Ap .I........ S.......GK .KE...A... ......I... .......... GK...D.S.. EFA....... .......... .......... ..........

AJ243438Pl .N.Y.S.S.. S.......RK DKG...A... ......I... .P........ .KK..D.S.. ELAA...... ..I....... ........P. ..........

D14996Ap R......S.. S......R-K .KG...TE.. T.....F... .A........ D.KAKD...A EPPI.S.... .......... .......... ....G.....

FN673831In .I.....S.. S......RNK .KG..RP..L T......... .A........ S.E.....GA Q..V.S.... .G.....G.. ........T. ....G.....

X99752Che E....R.F.. E...E.I.SR ...R...R.. T.....L... .T....S..E D.K..D.CGF ..TA...... ....CK.K.. ........P. ..........

EU223295Pe R--------- S.SIGRR.KK LETPEKPG.. T....L.SD. .A........ D.EGK..SHF EPAAE..QGV .GF..GGQSS LA........ ........K.

Consensus k.ehqgl.td s.fenflk.k g..kag.k.i t....svd#. ......f..# .q.ke#.ss. e.aakg.rrs esvpgrr.qt ps......s. ....v...e.

Resu

lts

188

401 460

AB326225Ap SDPGNLGADG VPGLGGGGQV NGGSEGGEFL QSEVSGGSHQ DLQNYIFGPE HKRDDFPSGL

NC_001409Pl .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

M58152Pl .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

AB326224Ap ..TR.VR... ...SD...E. .....SNRI. .F.G..QHD. S..D.L..S. .QQH...P.V

AJ243438Pl .N.R.V.... .SWTN...E. ......DRI. .F.GGRQPD. G..D.L.... YQQY.L.A.V

D14996Ap ..T.D..TNR ...RA...EI H......GVI ...GGSRFD. NI.D...... Y.QH.L.PSV

FN673831In ..SRD..T.. ..RRD..... H..P..DR.. .P.GG.QLDK ...D....S. Y.QH.L.AS.

X99752Che .SARDV..HR IL.PDS.... D....HLGVV .F.GG.EPD. S......... YQ.N...PS.

EU223295Pe RSSR....N. I.RR.SR.EE EW..HSAAEV ...DGR.AD. AFSDH...QK .QYPYL

Consensus sd.r#lgadg !pg..ggg#v .g.se....l ...ggg..dq .lq#yi..pe hqq.dlp...

(A)

Fig 4.22: (A) Multiple sequence alignment of complete MP isolates using Multalin program (Corpet, 1988). Amino acid sequences of MP

domains A (yellow) and domain B (green) are indicated. The conserved ‘D’ motif shown as * at amino acid positions 95/97 and 112/114 and

underlined amino acid residues in domains A and B are almost completely conserved within MPs belonging to the ‘30K superfamily. Consensus

levels: high=90% (in red font), low=50% (in blue font). Consensus symbols:! is anyone of IV, $ is anyone of LM, % is anyone of FY, # is

anyone of NDQEBZ. (B) Phylogenetic tree drawn with Tree Explorer ver. 2.12 (Tamura, 1999) showing relationship between complete MP

sequences from various host and countries. CMLV-MP used as root.

(B)

Resu

lts

189

1 * 100

NC_001409Pl MATMIRGHRL RIAEGDIPIA GVKSSRIYSD ISPFKKASDL MIHWNEFVFK VMPEDIAGDG FRLASIPVIP SSEVQAVLRK RESTNYVHWG ALSISIDALF

M58152Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AJ243438Pl ........K. K......... .......... V....R.... .......... .......... .....M.... .......... .......... ..........

AB326224Ap M.....K. .......... .......... .....R.... .......... ...D...... .......... .......... .......... ..........

FN673831In ..M....RK. Q..V...... ........E. V....R.... ..Q....... .......... .......... .......... ..G....... ..........

D14996Ap M.....K. K......... .......... V...R..... .......... .......... .......... .......... .......... ..........

AB326225Ap M.....K. K......... .......... V....R.... .......... .......... .......... D...K..I.. .D........ ..........

X99752Che ..L.....K. K......... .......... .....R.... .......... .......... .......... .T........ .......... ..........

EU223295Pe ..M.V...KS K......... ..R....... .T...R.... .......... ......V.K. .....V..V. I..I.S.... ..G....... ..........

AM888396In ..M.....K. K......... .......... V....R.... .......... .......... .......... .......... .......... ..........

AM888397In .....K..K. .........V .G........ V....R.... ....K..... .......... .......... .......... .......... ..........

Consensus mam.!r..Kl k........a .vk....... !s...r.... ....n..... ......a.d. .....!..!. s..!.a.... ..s....... ..........

101 * * * * 200

NC_001409Pl RKNAGVSGWC YVYDNRWETF EQAMLQKFHF NLDSGSATLV TSPNFPVSLD DPGLSNSISV AVMFENLNFK FESYPISVRV GNMCRFFDSF LSSVKNKVDS

M58152Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AJ243438Pl .......... .......... .......... .......... .......... .......... .......... L......... .......... ..........

AB326224Ap .......... .......... ...I....R. ........F. .......... .......... .......... L......... .....I...L ....R...E.

FN673831In .......... .......... ........R. .......... .......... .......... .......... L......... .......... ..........

D14996Ap .......... .......... ........R. .......... .......... .......... .......... L......... .......... ..C.......

AB326225Ap K......... .......... ...K....R. .......... .......... ........C. .......... LD........ .T........ ..N....N..

X99752Che .......... .......... .......... ........M. .......... .......... .......... L......... .......... ......R...

EU223295Pe K.....T.Q. ..F.K..T.. D..L....E. ...R.....I ....SS.... ....MD..C. .......... L.N....... .......... ....R..DE.

AM888396In .......... .......... ........R. .......... .

AM888397In .......... .......... ........R. ..N....... .

Consensus r.....s.w. ..%.n..e.. #..m....r. ..#s....$! .spn..vsld dpgl..si.v avmfenlnfk ...ypisvrv g.mcr.fds. ls.v.n...s

(A)

(B) Resu

lts

190

351 460

AB326225Ap NQAKENGGSS KSSTKGGRRS ESVPGRRRQT PSWKDRGNSG TDTGVHLREH SDPGNLGADG VPGLGGGGQV NGGSEGGEFL QSEVSGGSHQ DLQNYIFGPE HKRDDFPSGL

AB326224Ap GK...D.S.. EFA....... .......... .......... .......... ..TR.VR... ...SD...E. .....SNRI. .F.G..QHD. S..D.L..S. .QQH...P.V

AM494513In D.KE.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...EI ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM494510In D.KE.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...E. ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

FN666578In D.KE.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...E. ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM498046In D.K..D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...EI ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM498045In D.KA.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...EI ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

FN666579In D.KA.D.S.. ELTA.S.... .......... ........P. .......... ..AR.V.... I..SS..... ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM498048In D.KEKD.S.L E.T..S.... .......... ........P. .........Y .NTRDV.... I..SS...EI ......NRV. ...GG.QHD. S..D.L..S. .QQH...P.V

AJ243438Pl .KK..D.S.. ELAA...... ..I....... ........P. .......... .N.R.V.... .SWTN...E. ......DRI. .F.GGRQPD. G..D.L.... YQQY.L.A.V

AM494512In D.KE.D.SG. E.TI..R..F .GI...G... ........P. ..V....... ..TR.V..N. .S.SS...E. ...P.SDRVI .P.GG.EYD. G..D.....K .QQH..S..V


AM931533In D.KA.D.SG. E.TI..R..F .GI...G... ........P. ..V....... ..TR.V..N. .S.SS...E. ...P.SDRVI .P.GG.EYD. G..D.....K .QQH..S..V

AM494509In D.K..D.S.. E.T.Q.R..F KGI...G... ........P. ..V....... ..T.DV..N. ...S...SE. ...PKSDRVI .P.GG.QYD. G..D.....K .QQH...P.M

AM709777In D.KE.D.S.. E.T...R..F .GI...G... ........P. ..V....... ..TRDI..N. ...S...SE. ...PK.DRVI .P.GG.QYD. G..D.....K .QQH...P.V

AM498050In D.KE.D.S.. E.T..SR..F .GI...G... ........P. ..V....... ..TRDV..N. ...S...SE. ...PK.DRVI .P.GG.QYD. G..D.....K .QQH...P.V

AM494506In D.KE.D.S.. E.T...R..F .GI...G... ........P. ..V....... ..TR.VR... IS.S...SEI ......DRVI .P.GG.QHD. G..D....LK .QQY.L.P.V

D14996ApJa D.KAKD...A EPPI.S.... .......... .......... ....G..... ..T.D..TNR ...RA...EI H......GVI ...GGSRFD. NI.D...... Y.QH.L.PSV

FN550875In D.KE.D...A E..I...... .......... ........P. ....G..... ..T.D...N. ...RA...EI H..P...GVI ...GGSQ.N. .F.D...... ..QY.L.P.V

AM494507In D.KEKD...A E..V...... .......... ........P. ....G..... ..T.H...N. ...RA...EI H..P...GVI ...GG.QFN. ...D...... ..QY.L.P.V


AM494508In D.KE.D...A E..V...... .......... ........P. ....G..... ..T.H...N. ...RA...EI H..P...GVI ...GG.QFN. ...D...... ..QY.L.P.V



AM498049In D.KA.D...A E..V...... .......... ........P. ....G..... ..T.H...N. ...RA...EI H..P...GVI ...GG.QFN. ...D...... ..QY.L.P.V

AM408891In D.KE.D...A E..A...... .......... .......... ....G..... ..T.D.R.NR ...RDS..EI H..P...GVI ...GG.QFD. NI.D....H. ..QH.L.P.M

AM494511In D.KA.D...I E......... .......... .......... ....G..... ..T.D...NR I..RA...EI H..P...GVI ...GG.QLD. NI.D.F..S. ..QY.L.P.V

AM709776In D.KT.D...T ...A.S.... .......G.. ........P. ....G..... ..T.DF...R ...RA..SE. H...K..GVI ...GGSQLN. .I.D...... ..QY.L.P.V

AM409322In D.KE.D...A E....S.... .........I ........P. ....G..... ..T.D.R.NR ...RD...E. H..P...GVF ...GGSQFD. NI.D....H. ..

AM498047In D.KE.D...I E........F .G........ .......... ....G..... ..T.....N. I..RT...E. H..P...GVF ...GGSQFD. NI.D....H. ..

FN673831In S.E.....GA Q..V.S.... .G.....G.. ........T. ....G..... ..SRD..T.. ..RRD..... H..P..DR.. .P.GG.QLDK ...D....S. Y.QH.L.AS.

AM498044In D.KA.D...A E....S.... .......... ........T. ....G..... ...RD..... I.RRDS...I H..P..DRL. .P.GG.RFDK ...D....S. .QQH.L....

NC_001409 .....D.S.. EFAAQ...K. KGIS...K.. S.......P. .......... .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

M58152PlFr .....D.S.. EFAAQ...K. KGIS...K.. S.......P. .......... .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

X99752CheF D.K..D.CGF ..TA...... ....CK.K.. ........P. .......... .SARDV..HR IL.PDS.... D....HLGVV .F.GG.EPD. S......... YQ.N...PS.

FN550876In .......P. ....G..... ..T.H..... ...RA...EI H..P...GVI .P.GGSQFN. .F.D....S. ..QY.L.P.V

EU223295Pe D.EGK..SHF EPAAE..QGV .GF..GGQSS LA........ ........K. RSSR....N. I.RR.SR.EE EW..HSAAEV ...DGR.AD. AFSDH...QK .QYPYL

Consensus dqk.edg.s. es..kggrrs esvpgrrrqt ps......p. ..t.v...e. sdtg.lg.#g !pgr.ggg#. .g.seg..v. ...gggq.#. ..q#yi...e hqq.dlppg.

(C)

Resu

lts

191

Fig 4.23: Variability in MP isolates from India (grey shaded) and world shown using Multalin program (Corpet, 1988) (A) MP 5’-end [1-

200aa; also including 1-141 aa residues from partial MP isolates (AM888397 and AM888396) from India]. Amino acid sequences of MP

domains A (yellow) and domain B (green) are indicated. The conserved ‘D’ (95/97, 112/114, 149/151, 186/188) and ‘G’ (103/105) motifs shown

as * at amino acid positions and underlined amino acid residues in domains A and B are almost completely conserved within MPs belonging to

the ‘30K’ superfamily. (C) Multiple sequence alignment of MP 3’-end isolates [351-460aa]. Clustering of isolates in Group 1 (blue) and Group 2

clusters. Consensus levels: high=90% (in red font), low=50% (in blue font). Phylogenetic relationship of partial MP (B) of 5’ end [1-200aa] (D)

of 3’end [351-460aa] with distinct clustering in radiated tree. World isolates referred as Accession number, host, country initials, (isolate name)

and Indian isolates (red font) as Accession number, isolate name, host, (RC: Red Chief, Plp: Palampur, mp-movement protein).

(D)

Resu

lts

187

1 * 100

AB326225Ap MMIRGHKL KIAEGDIPIA GVKSSRIYSD VSPFKRASDL MIHWNEFVFK VMPEDIAGDG FRLASIPVIP DSEVKAVIRK RDSTNYVHWG ALSISIDALF

NC_001409Pl MAT.....R. R......... .......... I....K.... .......... .......... .......... S...Q..L.. .E........ ..........

M58152Pl MAT.....R. R......... .......... I....K.... .......... .......... .......... S...Q..L.. .E........ ..........

AB326224Ap ........ R......... .......... I......... .......... ...D...... .......... S...Q..L.. .E........ ..........

AJ243438Pl MAT....... .......... .......... .......... .......... .......... .....M.... S...Q..L.. .E........ ..........

D14996Ap ........ .......... .......... ....RK.... .......... .......... .......... S...Q..L.. .E........ ..........

FN673831In MA.....R.. Q..V...... ........E. .......... ..Q....... .......... .......... S...Q..L.. .EG....... ..........

X99752Che MAL....... .......... .......... I......... .......... .......... .......... ST..Q..L.. .E........ ..........

EU223295Pe MA..V....S .......... ..R....... IT........ .......... ......V.K. .....V..V. I..IQS.L.. .EG....... ..........

Consensus mam.!..hkl k..e...... ..k.....s. !s..kr.... ..h....... ...#..a.d. .....i..!. ss.!qa.l.. .#s....... ..........

101 * * * * 200

AB326225Ap KKNAGVSGWC YVYDNRWETF EQAKLQKFRF NLDSGSATLV TSPNFPVSLD DPGLSNSICV AVMFENLNFK LDSYPISVRV GTMCRFFDSF LSNVKNKNDS

NC_001409Pl R......... .......... ...M....H. .......... .......... ........S. .......... FE........ .N........ ..S....V..

M58152Pl R......... .......... ...M....H. .......... .......... ........S. .......... FE........ .N........ ..S....V..

AB326224Ap R......... .......... ...I...... ........F. .......... ........S. .......... .E........ .N...I...L ..S.R..VE.

AJ243438Pl R......... .......... ...M....H. .......... .......... ........S. .......... .E........ .N........ ..S....V..

D14996Ap R......... .......... ...M...... .......... .......... ........S. .......... .E........ .N........ ..C....V..

FN673831In R......... .......... ...M...... .......... .......... ........S. .......... .E........ .N........ ..S....V..

X99752Che R......... .......... ...M....H. ........M. .......... ........S. .......... .E........ .N........ ..S...RV..

EU223295Pe ......T.Q. ..F.K..T.. D..L....E. ...R.....I ....SS.... ....MD.... .......... .EN....... .N........ ..S.R..DE.

Consensus r.....s.w. ..%.n..e.. #..m...... ...s....l! ....fp.... ....s#..s. .......... l#s....... .n...f...f ..s.k.kv#.

201 300

AB326225Ap NFLLEASNAD PLGASVFGFD DDDQVSELFN YIQTVPTQAI KSREHEIPRG MFGIMGKKTV KSFEFASGSK GLNKIKPQRG RSSGRSSSQR FAPGFKSQDE

NC_001409Pl .......... ...VGA...E Q......... .......... .F......K. FL.M....KI .......... .MERR..N.. KQID..F... AV...R..N.

M58152Pl .......... ...VGA...E Q......... .......... .F......K. FL.M....KI .......... .MERR..N.. KQID..F... AV...R..N.

AB326224Ap ......A..E ....GA...E Q......... .......... ........K. L.......K. .T.......R SMERRR.... .PLE..T... VV........

AJ243438Pl ......A... ...VGA...E Q......... ..H....... ........K. L..ML...K. .........R SMGR...Y.. MQFE...... IV...R...D

D14996Ap ......A... ....GA...E Q......... .......... ......V... L..M....K. .........R N.GRR..... .PLE..A.L. V....R....

FN673831In ......A... ....GA...E Q......... .......... .......... L..M....K. .......... SMERRR.... .PLE...... VI........

X99752Che .....T.... ....GA...E N........D .......... ....L...K. F..ML...K. .......E.R NK..T..L.. .PAT..A... EL......AD

EU223295Pe .ARI..A..E ...LAD...E GG.RI....D .V.S..VM.V QTK.M...K. L..L...R.. .....T.KAG NARRREWSKL KI..DVAGVK -L.A.GGEF.

Consensus .fll.aa..# ...aga...# qd.q!....# .!qt..tq.! ksr.h.!.k. lf.m$..kk! .s...a.gs. ...rrkp.rg r...rs.sqr ...g..s#d#

301 400

AB326225Ap KVEHQGLATD LDFENFLKDE GRRKAGSKSI ASEGSSVDNI SSREFQFARQ NQAKENGGSS KSSTKGGRRS ESVPGRRRQT PSWKDRGNSG TDTGVHLREH

NC_001409Pl .......S.. S......RNK RGN...V..T .......... .......... .....D.S.. EFAAQ...K. KGIS...K.. S.......P. ..........

M58152Pl .......S.. S......RNK RGN...V..T .......... .......... .....D.S.. EFAAQ...K. KGIS...K.. S.......P. ..........

AB326224Ap .I........ S.......GK .KE...A... ......I... .......... GK...D.S.. EFA....... .......... .......... ..........

AJ243438Pl .N.Y.S.S.. S.......RK DKG...A... ......I... .P........ .KK..D.S.. ELAA...... ..I....... ........P. ..........

D14996Ap R......S.. S......R-K .KG...TE.. T.....F... .A........ D.KAKD...A EPPI.S.... .......... .......... ....G.....

FN673831In .I.....S.. S......RNK .KG..RP..L T......... .A........ S.E.....GA Q..V.S.... .G.....G.. ........T. ....G.....

X99752Che E....R.F.. E...E.I.SR ...R...R.. T.....L... .T....S..E D.K..D.CGF ..TA...... ....CK.K.. ........P. ..........

EU223295Pe R--------- S.SIGRR.KK LETPEKPG.. T....L.SD. .A........ D.EGK..SHF EPAAE..QGV .GF..GGQSS LA........ ........K.

Consensus k.ehqgl.td s.fenflk.k g..kag.k.i t....svd#. ......f..# .q.ke#.ss. e.aakg.rrs esvpgrr.qt ps......s. ....v...e.

Resu

lts

188

401 460

AB326225Ap SDPGNLGADG VPGLGGGGQV NGGSEGGEFL QSEVSGGSHQ DLQNYIFGPE HKRDDFPSGL

NC_001409Pl .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

M58152Pl .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

AB326224Ap ..TR.VR... ...SD...E. .....SNRI. .F.G..QHD. S..D.L..S. .QQH...P.V

AJ243438Pl .N.R.V.... .SWTN...E. ......DRI. .F.GGRQPD. G..D.L.... YQQY.L.A.V

D14996Ap ..T.D..TNR ...RA...EI H......GVI ...GGSRFD. NI.D...... Y.QH.L.PSV

FN673831In ..SRD..T.. ..RRD..... H..P..DR.. .P.GG.QLDK ...D....S. Y.QH.L.AS.

X99752Che .SARDV..HR IL.PDS.... D....HLGVV .F.GG.EPD. S......... YQ.N...PS.

EU223295Pe RSSR....N. I.RR.SR.EE EW..HSAAEV ...DGR.AD. AFSDH...QK .QYPYL

Consensus sd.r#lgadg !pg..ggg#v .g.se....l ...ggg..dq .lq#yi..pe hqq.dlp...

(A)

Fig 4.22: (A) Multiple sequence alignment of complete MP isolates using Multalin program (Corpet, 1988). Amino acid sequences of MP

domains A (yellow) and domain B (green) are indicated. The conserved ‘D’ motif shown as * at amino acid positions 95/97 and 112/114 and

underlined amino acid residues in domains A and B are almost completely conserved within MPs belonging to the ‘30K superfamily. Consensus

levels: high=90% (in red font), low=50% (in blue font). Consensus symbols:! is anyone of IV, $ is anyone of LM, % is anyone of FY, # is

anyone of NDQEBZ. (B) Phylogenetic tree drawn with Tree Explorer ver. 2.12 (Tamura, 1999) showing relationship between complete MP

sequences from various host and countries. CMLV-MP used as root.

(B)

Resu

lts

189

1 * 100

NC_001409Pl MATMIRGHRL RIAEGDIPIA GVKSSRIYSD ISPFKKASDL MIHWNEFVFK VMPEDIAGDG FRLASIPVIP SSEVQAVLRK RESTNYVHWG ALSISIDALF

M58152Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AJ243438Pl ........K. K......... .......... V....R.... .......... .......... .....M.... .......... .......... ..........

AB326224Ap M.....K. .......... .......... .....R.... .......... ...D...... .......... .......... .......... ..........

FN673831In ..M....RK. Q..V...... ........E. V....R.... ..Q....... .......... .......... .......... ..G....... ..........

D14996Ap M.....K. K......... .......... V...R..... .......... .......... .......... .......... .......... ..........

AB326225Ap M.....K. K......... .......... V....R.... .......... .......... .......... D...K..I.. .D........ ..........

X99752Che ..L.....K. K......... .......... .....R.... .......... .......... .......... .T........ .......... ..........

EU223295Pe ..M.V...KS K......... ..R....... .T...R.... .......... ......V.K. .....V..V. I..I.S.... ..G....... ..........

AM888396In ..M.....K. K......... .......... V....R.... .......... .......... .......... .......... .......... ..........

AM888397In .....K..K. .........V .G........ V....R.... ....K..... .......... .......... .......... .......... ..........

Consensus mam.!r..Kl k........a .vk....... !s...r.... ....n..... ......a.d. .....!..!. s..!.a.... ..s....... ..........

101 * * * * 200

NC_001409Pl RKNAGVSGWC YVYDNRWETF EQAMLQKFHF NLDSGSATLV TSPNFPVSLD DPGLSNSISV AVMFENLNFK FESYPISVRV GNMCRFFDSF LSSVKNKVDS

M58152Pl .......... .......... .......... .......... .......... .......... .......... .......... .......... ..........

AJ243438Pl .......... .......... .......... .......... .......... .......... .......... L......... .......... ..........

AB326224Ap .......... .......... ...I....R. ........F. .......... .......... .......... L......... .....I...L ....R...E.

FN673831In .......... .......... ........R. .......... .......... .......... .......... L......... .......... ..........

D14996Ap .......... .......... ........R. .......... .......... .......... .......... L......... .......... ..C.......

AB326225Ap K......... .......... ...K....R. .......... .......... ........C. .......... LD........ .T........ ..N....N..

X99752Che .......... .......... .......... ........M. .......... .......... .......... L......... .......... ......R...

EU223295Pe K.....T.Q. ..F.K..T.. D..L....E. ...R.....I ....SS.... ....MD..C. .......... L.N....... .......... ....R..DE.

AM888396In .......... .......... ........R. .......... .

AM888397In .......... .......... ........R. ..N....... .

Consensus r.....s.w. ..%.n..e.. #..m....r. ..#s....$! .spn..vsld dpgl..si.v avmfenlnfk ...ypisvrv g.mcr.fds. ls.v.n...s

(A)

(B) Resu

lts

190

351 460

AB326225Ap NQAKENGGSS KSSTKGGRRS ESVPGRRRQT PSWKDRGNSG TDTGVHLREH SDPGNLGADG VPGLGGGGQV NGGSEGGEFL QSEVSGGSHQ DLQNYIFGPE HKRDDFPSGL

AB326224Ap GK...D.S.. EFA....... .......... .......... .......... ..TR.VR... ...SD...E. .....SNRI. .F.G..QHD. S..D.L..S. .QQH...P.V

AM494513In D.KE.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...EI ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM494510In D.KE.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...E. ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

FN666578In D.KE.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...E. ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM498046In D.K..D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...EI ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM498045In D.KA.D.S.. ELTA.S.... .......... ........P. .......... ..TR.V.... I..SS...EI ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

FN666579In D.KA.D.S.. ELTA.S.... .......... ........P. .......... ..AR.V.... I..SS..... ...P..NRV. .F.GG.QHD. SF.D.L..S. .QQH...P.V

AM498048In D.KEKD.S.L E.T..S.... .......... ........P. .........Y .NTRDV.... I..SS...EI ......NRV. ...GG.QHD. S..D.L..S. .QQH...P.V

AJ243438Pl .KK..D.S.. ELAA...... ..I....... ........P. .......... .N.R.V.... .SWTN...E. ......DRI. .F.GGRQPD. G..D.L.... YQQY.L.A.V



AM931533In D.KA.D.SG. E.TI..R..F .GI...G... ........P. ..V....... ..TR.V..N. .S.SS...E. ...P.SDRVI .P.GG.EYD. G..D.....K .QQH..S..V

AM494509In D.K..D.S.. E.T.Q.R..F KGI...G... ........P. ..V....... ..T.DV..N. ...S...SE. ...PKSDRVI .P.GG.QYD. G..D.....K .QQH...P.M

AM709777In D.KE.D.S.. E.T...R..F .GI...G... ........P. ..V....... ..TRDI..N. ...S...SE. ...PK.DRVI .P.GG.QYD. G..D.....K .QQH...P.V

AM498050In D.KE.D.S.. E.T..SR..F .GI...G... ........P. ..V....... ..TRDV..N. ...S...SE. ...PK.DRVI .P.GG.QYD. G..D.....K .QQH...P.V

AM494506In D.KE.D.S.. E.T...R..F .GI...G... ........P. ..V....... ..TR.VR... IS.S...SEI ......DRVI .P.GG.QHD. G..D....LK .QQY.L.P.V

D14996ApJa D.KAKD...A EPPI.S.... .......... .......... ....G..... ..T.D..TNR ...RA...EI H......GVI ...GGSRFD. NI.D...... Y.QH.L.PSV

FN550875In D.KE.D...A E..I...... .......... ........P. ....G..... ..T.D...N. ...RA...EI H..P...GVI ...GGSQ.N. .F.D...... ..QY.L.P.V






AM498049In D.KA.D...A E..V...... .......... ........P. ....G..... ..T.H...N. ...RA...EI H..P...GVI ...GG.QFN. ...D...... ..QY.L.P.V

AM408891In D.KE.D...A E..A...... .......... .......... ....G..... ..T.D.R.NR ...RDS..EI H..P...GVI ...GG.QFD. NI.D....H. ..QH.L.P.M

AM494511In D.KA.D...I E......... .......... .......... ....G..... ..T.D...NR I..RA...EI H..P...GVI ...GG.QLD. NI.D.F..S. ..QY.L.P.V

AM709776In D.KT.D...T ...A.S.... .......G.. ........P. ....G..... ..T.DF...R ...RA..SE. H...K..GVI ...GGSQLN. .I.D...... ..QY.L.P.V

AM409322In D.KE.D...A E....S.... .........I ........P. ....G..... ..T.D.R.NR ...RD...E. H..P...GVF ...GGSQFD. NI.D....H. ..

AM498047In D.KE.D...I E........F .G........ .......... ....G..... ..T.....N. I..RT...E. H..P...GVF ...GGSQFD. NI.D....H. ..

FN673831In S.E.....GA Q..V.S.... .G.....G.. ........T. ....G..... ..SRD..T.. ..RRD..... H..P..DR.. .P.GG.QLDK ...D....S. Y.QH.L.AS.

AM498044In D.KA.D...A E....S.... .......... ........T. ....G..... ...RD..... I.RRDS...I H..P..DRL. .P.GG.RFDK ...D....S. .QQH.L....

NC_001409 .....D.S.. EFAAQ...K. KGIS...K.. S.......P. .......... .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

M58152PlFr .....D.S.. EFAAQ...K. KGIS...K.. S.......P. .......... .....VR... .S.PS..SEI ....ISPRV. .P.G..QLD. SF.D.L.... .QQN.I....

X99752CheF D.K..D.CGF ..TA...... ....CK.K.. ........P. .......... .SARDV..HR IL.PDS.... D....HLGVV .F.GG.EPD. S......... YQ.N...PS.

FN550876In .......P. ....G..... ..T.H..... ...RA...EI H..P...GVI .P.GGSQFN. .F.D....S. ..QY.L.P.V

EU223295Pe D.EGK..SHF EPAAE..QGV .GF..GGQSS LA........ ........K. RSSR....N. I.RR.SR.EE EW..HSAAEV ...DGR.AD. AFSDH...QK .QYPYL

Consensus dqk.edg.s. es..kggrrs esvpgrrrqt ps......p. ..t.v...e. sdtg.lg.#g !pgr.ggg#. .g.seg..v. ...gggq.#. ..q#yi...e hqq.dlppg.

(C)

Resu

lts

191

Fig 4.23: Variability in MP isolates from India (grey shaded) and world shown using Multalin program (Corpet, 1988) (A) MP 5’-end [1-

200aa; also including 1-141 aa residues from partial MP isolates (AM888397 and AM888396) from India]. Amino acid sequences of MP

domains A (yellow) and domain B (green) are indicated. The conserved ‘D’ (95/97, 112/114, 149/151, 186/188) and ‘G’ (103/105) motifs shown

as * at amino acid positions and underlined amino acid residues in domains A and B are almost completely conserved within MPs belonging to

the ‘30K’ superfamily. (C) Multiple sequence alignment of MP 3’-end isolates [351-460aa]. Clustering of isolates in Group 1 (blue) and Group 2

clusters. Consensus levels: high=90% (in red font), low=50% (in blue font). Phylogenetic relationship of partial MP (B) of 5’ end [1-200aa] (D)

of 3’end [351-460aa] with distinct clustering in radiated tree. World isolates referred as Accession number, host, country initials, (isolate name)

and Indian isolates (red font) as Accession number, isolate name, host, (RC: Red Chief, Plp: Palampur, mp-movement protein).

(D)

Resu

lts

192

Fig 4.24: MaxChi plot showing recombination between FN673831India15Apmp (major parent, apple) and EU223295 (TaTao-5 peach

isolate, minor parent) and formation of daughter isolate AB326225 (MO-5, apple) using RDP ver. 1.08 (Martin et al., 2005)

Resu

lts

Results

193

4.5.3 Sequence, Phylogenetic and Recombination Analysis of ACLSV-

Replicase Gene

Sequencing of replicase amplified in two parts from India15 (J&K) isolate revealed

that first was ~1572bp (from 5’- 28nt to 1580nt - 3’) and the second from middle

portion ~420bp (from 5’- 4502nt to 4922 nt - 3’). The replicase C-terminal end

overlapping the MP N-terminal (~67nt) was deduced from three amplicons (Table

4.24) obtained using primer combination 5700(MPup)-6112dn yielding ~444bp. The

region was translated to 36aa of replicase gene from the 3’-end. The replicase was

finally analyzed in three parts (5’-end, middle portion and 3’-end) with 8 sequences

listed in Table 4.24.

Three sequences from the 5’-end of ACLSV genome were used for analysis of

replicase gene. The putative methyltransferase (MET) domain was located in the N-

terminal region (43-336 amino acids, underlined) was mostly conserved (Fig 4.27

A). The MET domain of Indian isolates showed 15 amino acid changes highlighted

in yellow. Most of these were conserved however, six unique changes viz. at 59th

position E (Glutamic Acid, acidic) to G (Glycine, polar), 165 D (Aspartic Acid,

acidic) to S (Serine, polar), 278 Y (Tyrosine, polar) to F (Phenylalanine,

hydrophobic), 381 R (Arginine, basic) to G, 443 K (Lysine, basic) to E and 481 G to

D were present (Fig 4.27 A). The Indian isolates FN826785 (India15ApRep2) and

FN826786 (India15ApRep3) shared sequence identity of 98% at amino acid (aa)

level, while showing 93 and 92 percent identity to India15ApRep1 (FN826784)

isolate respectively. The last 86aa had maximum variability. The India15ApRep1

(FN826784) isolate was maximally (96%) similar at aa level to P-205 isolate. The

other two Indian isolates shared maximum (94%) similarity to B6 isolate (Table 4.25

A). Phylogenetically TaTao-5 isolate was farthest from the rest of the isolates. All

the Indian isolates clustered together with other apple isolates while stone fruit

isolates clearly showed difference in evolution. The PBM-1 isolate (AJ24438) from

plum was an exception, as shared greater sequence identity to apple isolates (Table

4.25 A) and also clustered among them (Fig 4.27 B).

Sequence analysis of middle part of ACLSV-Replicase gene (from 1466 to 1588aa-

123aa) indicated low variability (Fig 4.27 C) and sharing 91-95% identity at aa level

with all the isolates used in the study irrespective of isolation host (Table 4.25 B). No

host-wise clustering was noted in phylogenetic tree (Fig 4.27 E). However, the

Results

194

Indian isolate showed closer phylogeny to stone fruit isolates even after sharing same

sequence identity (95% at aa level) with P-205 apple isolate (Fig 4.27 E). The unique

aa changes in Indian sequence FN555394 (India15ApRepMid) viz. T (Threonine,

polar) to P (Proline, hydrophobic), S (polar) to A (Alanine, hydrophobic) and

K(basic) to L (Leucine, hydrophobic). Similarly the 3’-end portion of ACLSV-

Replicase (~1859-1899-36aa) was much conserved with only four unique changes in

the Indian isolates viz. E (acidic) to S (polar), W (Tryptophan, hydrophobic) to G

(polar), S (polar) to L (hydrophobic), D (Aspartic Acid, acidic) to T (polar) and E

(acidic) to A (hydrophobic) are highlighted and stared (Fig 4.27 D). The Indian

isolates India15ApRep3’ (FN673831) and IndiaApRep3’RC-Plp (AM888397) shared

88% sequence identity at amino acid level and were least similar (68-83%) to rest of

the world isolates. These isolate shared 74 and 82 % similarity with India11ApRep3’

(AM888396) isolate respectively. The India11ApRep3’ isolate was 100% identical at

aa level to plum PBM-1(AJ24438) isolate and both these isolates shared maximum

identity to TaTao-5 peach isolate (Table 4.25 C) and cluster together. The clustering

in phylogenetic tree shows difference in evolution of two of the Indian isolates (Fig

4.27 F).

Recombination analysis using RDP ver 1.08 for all the three regions of replicase

amplified in the study have been enlisted in Table 4.26. Maximum recombination

events with significant average P-values were detected from 5’-end ACLSV-

Replicase sequences. The last 130nt region (downstream the MET domain) came

across as the recombination target. Five different recombination events yielding

daughter ACLSV plum isolates P-863 (M58152) or NC_001409 were detected by

five recombination programmes each. One such event with FN826785

India15ApRep2 (major parent, apple) and AB326225 (MO-5 plum isolate, minor

parent) having break points at 1472-1569nt gave daughter isolate NC_001409

(Plum). The event is represented as MaxChi plot with clear cut breakpoints (Fig

4.28). No significant events of recombination were detected from middle

(downstream helicase domain) however, one PRE from polymerase downstream

region at the 3’-end of replicase including a few nucleotides from the Replicase-MP

overlapping region was observed.

Results

195

Table 4.24: All the Replicase sequences used for analyses

S. no. Accession

no. Country/Region Isolate name

Part

analyzed Source

1. EU223295 USA TaTao5

5’-end,

3’-end and

middle

peach (Pe)

2. D14996 Japan (Jap) P-205 -do- apple (Ap)

3. AB326225 Japan (Jap) MO-5 -do- apple (Ap)

4. AB326224 Japan (Jap) B6 -do- apple (Ap)

5. M58152 France (Fra) P863 -do- plum (Pl)

6. NP_040553 France (Fra) - -do- plum (Pl)

7. AJ243438 Germany (Ger) PBM1 -do- plum (Pl)

8. X99752 France (Fra) Balton-1 -do-

sweet

cherry

(Che)

9. FN826784 India (Kashmir) India15ApRep1 5’end apple (Ap)



12. FN555394 India (Kashmir) India15ApRepMid middle apple (Ap)

13. FN673831 India (Kashmir) India15ApRep3’ 3’end apple (Ap)

14. AM888397 Palampur (HP) IndiaApRep3’

(RC-Plp) 3’end apple (Ap)

15. AM888396 Kinnaur (HP) India11ApRep3’ 3’end apple (Ap)

Results

193

4.5.3 Sequence, Phylogenetic and Recombination Analysis of ACLSV-

Replicase Gene

Sequencing of replicase amplified in two parts from India15 (J&K) isolate revealed

that first was ~1572bp (from 5’- 28nt to 1580nt - 3’) and the second from middle

portion ~420bp (from 5’- 4502nt to 4922 nt - 3’). The replicase C-terminal end

overlapping the MP N-terminal (~67nt) was deduced from three amplicons (Table

4.24) obtained using primer combination 5700(MPup)-6112dn yielding ~444bp. The

region was translated to 36aa of replicase gene from the 3’-end. The replicase was

finally analyzed in three parts (5’-end, middle portion and 3’-end) with 8 sequences

listed in Table 4.24.

Three sequences from the 5’-end of ACLSV genome were used for analysis of

replicase gene. The putative methyltransferase (MET) domain was located in the N-

terminal region (43-336 amino acids, underlined) was mostly conserved (Fig 4.27

A). The MET domain of Indian isolates showed 15 amino acid changes highlighted

in yellow. Most of these were conserved however, six unique changes viz. at 59th

position E (Glutamic Acid, acidic) to G (Glycine, polar), 165 D (Aspartic Acid,

acidic) to S (Serine, polar), 278 Y (Tyrosine, polar) to F (Phenylalanine,

hydrophobic), 381 R (Arginine, basic) to G, 443 K (Lysine, basic) to E and 481 G to

D were present (Fig 4.27 A). The Indian isolates FN826785 (India15ApRep2) and

FN826786 (India15ApRep3) shared sequence identity of 98% at amino acid (aa)

level, while showing 93 and 92 percent identity to India15ApRep1 (FN826784)

isolate respectively. The last 86aa had maximum variability. The India15ApRep1

(FN826784) isolate was maximally (96%) similar at aa level to P-205 isolate. The

other two Indian isolates shared maximum (94%) similarity to B6 isolate (Table 4.25

A). Phylogenetically TaTao-5 isolate was farthest from the rest of the isolates. All

the Indian isolates clustered together with other apple isolates while stone fruit

isolates clearly showed difference in evolution. The PBM-1 isolate (AJ24438) from

plum was an exception, as shared greater sequence identity to apple isolates (Table

4.25 A) and also clustered among them (Fig 4.27 B).

Sequence analysis of middle part of ACLSV-Replicase gene (from 1466 to 1588aa-

123aa) indicated low variability (Fig 4.27 C) and sharing 91-95% identity at aa level

with all the isolates used in the study irrespective of isolation host (Table 4.25 B). No

host-wise clustering was noted in phylogenetic tree (Fig 4.27 E). However, the

Results

194

Indian isolate showed closer phylogeny to stone fruit isolates even after sharing same

sequence identity (95% at aa level) with P-205 apple isolate (Fig 4.27 E). The unique

aa changes in Indian sequence FN555394 (India15ApRepMid) viz. T (Threonine,

polar) to P (Proline, hydrophobic), S (polar) to A (Alanine, hydrophobic) and

K(basic) to L (Leucine, hydrophobic). Similarly the 3’-end portion of ACLSV-

Replicase (~1859-1899-36aa) was much conserved with only four unique changes in

the Indian isolates viz. E (acidic) to S (polar), W (Tryptophan, hydrophobic) to G

(polar), S (polar) to L (hydrophobic), D (Aspartic Acid, acidic) to T (polar) and E

(acidic) to A (hydrophobic) are highlighted and stared (Fig 4.27 D). The Indian

isolates India15ApRep3’ (FN673831) and IndiaApRep3’RC-Plp (AM888397) shared

88% sequence identity at amino acid level and were least similar (68-83%) to rest of

the world isolates. These isolate shared 74 and 82 % similarity with India11ApRep3’

(AM888396) isolate respectively. The India11ApRep3’ isolate was 100% identical at

aa level to plum PBM-1(AJ24438) isolate and both these isolates shared maximum

identity to TaTao-5 peach isolate (Table 4.25 C) and cluster together. The clustering

in phylogenetic tree shows difference in evolution of two of the Indian isolates (Fig

4.27 F).

Recombination analysis using RDP ver 1.08 for all the three regions of replicase

amplified in the study have been enlisted in Table 4.26. Maximum recombination

events with significant average P-values were detected from 5’-end ACLSV-

Replicase sequences. The last 130nt region (downstream the MET domain) came

across as the recombination target. Five different recombination events yielding

daughter ACLSV plum isolates P-863 (M58152) or NC_001409 were detected by

five recombination programmes each. One such event with FN826785

India15ApRep2 (major parent, apple) and AB326225 (MO-5 plum isolate, minor

parent) having break points at 1472-1569nt gave daughter isolate NC_001409

(Plum). The event is represented as MaxChi plot with clear cut breakpoints (Fig

4.28). No significant events of recombination were detected from middle

(downstream helicase domain) however, one PRE from polymerase downstream

region at the 3’-end of replicase including a few nucleotides from the Replicase-MP

overlapping region was observed.

Results

195

Table 4.24: All the Replicase sequences used for analyses

S. no. Accession

no. Country/Region Isolate name

Part

analyzed Source

1. EU223295 USA TaTao5

5’-end,

3’-end and

middle

peach (Pe)

2. D14996 Japan (Jap) P-205 -do- apple (Ap)

3. AB326225 Japan (Jap) MO-5 -do- apple (Ap)

4. AB326224 Japan (Jap) B6 -do- apple (Ap)

5. M58152 France (Fra) P863 -do- plum (Pl)

6. NP_040553 France (Fra) - -do- plum (Pl)

7. AJ243438 Germany (Ger) PBM1 -do- plum (Pl)

8. X99752 France (Fra) Balton-1 -do-

sweet

cherry

(Che)




12. FN555394 India (Kashmir) India15ApRepMid middle apple (Ap)

13. FN673831 India (Kashmir) India15ApRep3’ 3’end apple (Ap)

14. AM888397 Palampur (HP) IndiaApRep3’

(RC-Plp) 3’end apple (Ap)

15. AM888396 Kinnaur (HP) India11ApRep3’ 3’end apple (Ap)

196

1 * 100

AB326224Ap MAFSYRTPQE ELLXRLPQSQ QEAISGFQYE RIQKEEEKKV ENFSFYLPEK TREWFTKSGV YLSPFAYVNH SHPGCKTLEN HLLFNVVSSY ISKYSYVACL

D14996ApJa .......... ...S...... .......... .F........ .......... .......... .......... .......... .......A.. ..........

FN826784Ap .......... ...S...... .......... ...E...... .......... .......... .......... .......... .......A.. ..........

AJ243438Pl .......... ...S...... ..V....... .......... ..LF...... .......... .......... .......... .......A.. ..........

FN826785Ap .......... ...S...... .......... .......... ........GR .......... .......... .......... .......A.. ..........

FN826786Ap .......... ...S...... .......... .......... .........R .......... .......... .......... .......A.. ..........

M58152PlFr .......... ...S...... ..V....... .......... .......... .......... .......... .......... .......A.. ..........

NC_001409P .......... ...S...... ..V....... .......... .......... .......... .......... .......... .......A.. ..........

X99752CheF .......... ...S....A. ..V.....F. .......... .......... .......... .......... .......... .......A.. ..........

AB326225Ap .......... ...S...... ..V...Y... .......... .........R .K........ .......... .......... .......A.. ....P.....

EU223295Pe .......... ...N...... ..ILGK..F. ..E....... A...YF.... .......... .......ET. .......... .......A.. ....P.....

Consensus .......... ...s....s. ...isg%.%. ..#....... e...%%...k .r........ .......vn. .......... .......A.. ....s.....

101 * 200

AB326224Ap SIKSNKMSKM ERLGSNSVKT YDILNRLVTA KDKARYGPLA KPERSPCPRK TNIFIHDEIH YWSRDQLETF LQVHRPKNLW ATLVFPPEIL AGYKSSVLPF

D14996ApJa .......... .......... .......... .......... R.......K. .......... .......... .......... .......... ..........

FN826784Ap .......... .......... .......... .......... ........K. .......... .......... ...Y...... .......... ..........

AJ243438Pl .......... .......... .........V .G.G...... ........K. .......... .......... .......... .......... ..........

FN826785Ap .......... .......... .........V .......... R.......K. .......... ....S..... ...YK..... .......... ..........

FN826786Ap .....R.... .......... .........V .......... R.......K. .......... ....S..... ...YK..... ........V. ..........

M58152PlFr .......... ....P..... .......... .......... ........K. .......... .......... .......... .......... ..........

NC_001409P .......... ....P..... .......... .......... ........K. .......... .......... .......... .......... ..........

X99752CheF ...A...... ....P..... ..V....... .......... ....A...K. .......... ........S. ...YK..... .......... ...R......

AB326225Ap .......... ....P...R. .........S .......... T.......K. .......... ....T...S. .M........ .......... ..........

EU223295Pe .......... ....AS.... .......... .........V SEV.A...K. .......... ....K...N. .MINK..... .......... ...R......

Consensus ...s...... .....n..k. ..!......a .........a .pe.s...K. .......... ....d...t. .q!.r..... .......... ...k......

201 * 300

AB326224Ap LYQFEIHGKD LVYMPDGVRS ESYTQPLENG FLLSSSSILT RNKTTGVELR YQVSLVYSLG SHHLFHIYPT EDLMKEEVRR FGPYDLFDVG SLFVKPVRVP

D14996ApJa .......... .......... .......... .........V ...A....I. .......... .......... .......... .......... ..........

FN826784Ap ........R. .......... .......... .....G...V ........M. .......... .......F.. .......... .......... T.........

AJ243438Pl .......... .......... .......... Y........V ........M. .....I.... .......... .......... .......... ..........

FN826785Ap ........E. .......... .......... .........V ......I.M. .......... .......... .......... .......... ..........

FN826786Ap .......... .......... .......... .........V ......I.M. .......... .......... .......... .......... ..........

M58152PlFr .......... .......... .......... ........II K.RV....I. .......... .........A .......... .......... ..........

NC_001409P .......... .......... .......... ........II K.RV....I. .......... .........A .......... .......... ..........

X99752CheF .......... .I........ .......... ........II K..ND.S... .....I.... ......M... .N.L...... .......... ..........

AB326225Ap .......... .......... .......... Y........V H..K...... .......... .......F.. ...L...... .......... ..........

EU223295Pe ......S... .I........ .......... Y....N..II FDHCKKK.I. ..I..I.... .......F.C Q......... .......... ....R.....

Consensus ......h.k. .!........ .......... %....s..l! .#k.tgv... ..!..!.... ......i%.t ##.$...... .......... ....k.....

Resu

lts

197

301 * 400

AB326224Ap IQDFPLSVFK KIFIYLSSLK KPDVQSAVAK LRQLPDADIS IESVFMVQEF ASRVEKNGVG NWSCSFWECM KDWFFDKLPY REVLERIGLA DDLTRRLMKI

D14996ApJa .......... .......... .......... ....S..... .......... .......... S......... .......... .....K.... N.F.......

FN826784Ap .......... .......... R......... ....S..... .......... .......... .......... .......... .....K.... ..F.......

AJ243438Pl .......... .....I.... R.G....... ....S..... .......... ...I..D..E K......G.. .......... .....K.... ..F.......

FN826785Ap .......... .......... .......... ....S..... .......... .......... S......... ......R... G....K.... ..F.......

FN826786Ap .......... .......... .......... ....S..... .......... .......... S......G.. .......... G....K.... ..F.......

M58152PlFr .......... .....M.... .......... ....S..... ......I... ...I.....E S......G.. .......... .....K.... ..F.......

NC_001409P .......... .....M.... .......... ....S..... ......I... ...I.....E S......G.. .......... .....K.... ..F.......

X99752CheF .......... .......... .......... ....S..... .......... ...I..E.IE G......G.. .......... .....K.... ..F.......

AB326225Ap .......... .......... .......... ....S..... .......... ...I...... S......... .......... ...I.KV... ..F......L

EU223295Pe .......... .......... ...E...... ....S..... .....L.... ...I..H.L. .......D.. .......... NH...K.... ..F......L

Consensus .......... .....$.... k..v...... ....S..... .....$!... ...!..n.vg .......... .......... re.l.K!... ..F......i

401 * * 486

AB326224Ap KPLAFDIHTT DRPLTVRMVI DQIWEERQSS CDDIPNIVFY GRKEWLEHGV MPKVKK-GLA KLVPGREFDS GDYPKEIYSD LLSSTS

D14996ApJa .......... .......... ....G..... ...V...I.. .........L L.....-... .......TGG S...E..... ......

FN826784Ap .......... .......... ....G..... ...V...I.. .......N.L L.....-... .......IGG S.C.E..... .....

AJ243438Pl .......... .....D.... .......... .......... .....I.... V..I..-S.. ..I....ME. S......... ......

FN826785Ap .......Y.. .......... ..V....... S......... .........I ...M.R-... .......P.. D......... .....

FN826786Ap .......Y.. .......... ..V....... S......... ..E......I ...M.R-... .......P.. D......... .....

M58152PlFr .......... ........I. .......L.. F.N.S..... ..R...NN.. L.....K... ..I....V.. HN..R..... ......

NC_001409P .......... ........I. .......L.. F.N.S..... ..R...NN.. L.....K... ..I....V.. HN..R..... ......

X99752CheF .........S .......... .......C.V N...S..I.. ..N..M...I V.RI..-.M. ..INRK.ALP SR..V..... ......

AB326225Ap .........S .......... .S.....VCM E.SL..QI.. .....IQY.. ..MI.R-.FS ......DMLE PS..Q..F.. .....P

EU223295Pe ...S.....S .Q........ .R..GPD.IE D.PLVECISK D.IGVIHNNC IVRGIQ-.VK TILKLDSSKV HV.NY..... I.AT.P

Consensus ...a...h.t .r......!. .q!.eerqss ..dip#i!fy g..ew.e.g. .pk.kk..la kl.pgre... ...p...%.. l.ss..

(A)

(B)

Resu

lts

198

1466 * * 1545

AB326224Ap RMDDDLTF LAAIKKRLRF DNVANNYAKF KAAENKGKYL AKVFLKHVPI KCGRDQRLLD QCRQEFEETK LSKSAATIGA HSQRSDSDWP LDKIFLFMKS

M58152PlFr RHK....... .......... .......... ....SR.... T.I....... .......... .......... .......... .......... ..........

NC_001409P RHK....... .......... .......... ....SR.... T.I....... .......... .......... .......... .......... ..........

X99752CheF RHK....... .......... .......S.. ....SR.... ..I....... .......... .......... .......... .......... ..........

D14996ApJa RH........ .......... .......... ....SR.... .....R.... .......... .......... .......... .......... ..........

AJ243438Pl RH........ .......... .......... .....R.... ..I....... .......... .......... .......... .......... ..........

AB326225Ap RHK....... .......... .........L ..S..R.... .......... .......... .......... .......... .A........ ..........

FN555394In RHK....... .......... .......... ....SR.... .......... .......... .......... ......P... ......A... ..........

EU223295Pe RH........ .......... .S....M... ....SR.... ..I....... .S........ A......... .......... ......T... ..........

Consensus rhr....... .......... .n....y..f ..a.sr.... ..!....... .c........ q......... ......t... .s....s... ..........

1546 * 1588

AB326224Ap QLCTKFEKRF TEAKAGQTLA CFP

M58152PlFr .......... .......... ...

NC_001409P .......... .......... ...

X99752CheF .......... .......... ...

D14996ApJa .......... .......... ...

AJ243438Pl .......... .......... ...

AB326225Ap .......... .......... ...

FN555394In ....LL.... .......... ...

EU223295Pe .......... .......... ...

Consensus ....kf.... .......... ...

(C)

1859 ** ** * 1899

AB326224Ap LEWLSDEDGD DDKGSQIENR RRGYSNCWGE KLQNLF

AB326225Ap .......... ......V.D. .......... ......

AJ243438Pl .......... ........D. .......... ......

M58152PlFr .......... N.......D. .......... ......

NC_001409P .......... N.......D. .......... ......

D14996ApJa ........S. ........D. .......... ......

AM888396In ......... ........D. .......... ......

X99752CheF .........I ......V.D. .......... ......

EU223295Pe .......... .GQ....KD. ....T..... ......

FN673831In .SGFLT.... .....K.AD. S......... .....

AM888397In .SGFLT.... .......... .......... ......

Consensus .ewlsd...d .dk..q!e#. r...s..... .....f

(D)

Resu

lts

199

(E) (F)

Fig 4.27: Multiple sequence alignment of ACLSV-replicase gene sequences at amino acid level using Multalin program (Corpet, 1998).

(A) 486 aa from 5’end. The conserved methyl-transferase domain is underlined. (C) Middle portion (1466-1588 aa) of replicase gene. (D) 3’end

(1859-1899 aa; 36aa overlapping region of Replicase-MP gene). All the Indian isolates are shaded (grey) and the changes in aa residues are

highlighted in yellow. Phylogenetic relationship on the basis of replicase gene sequences at amino acid level of all the ACLSV isolates (Indian

and world) used in the study. “*” denotes unique changes in amino acids. (B) Phylogenetic tree drawn by Neighbor Joining method using MEGA

version 4 (Tamura et al., 2007) with 50% bootstrap cut-off value for 5’end. Phylogenetic tree (E) Drawn with Tree Explorer ver. 2.12 (Tamura,

1999) showing relationship for middle portion (F) Drawn drawn by Neighbor Joining method using MEGA version 4 (Tamura et al., 2007) with

50% bootstrap cut-off value for 3’end of replicase gene.

Resu

lts

200

Table 4.25(A): Percentage sequence identities of complete ACLSV-Replicase 5’end region at amino acid level (below diagonal) among

different ACLSV isolates using ClustalW.

Ta

Tao -5 B6 P863 Pl P-205 Bal-1 MO-5 PBM1 Rep1 Rep2 Rep3

EU223295 (TaTao 5) Pe x

AB326224 (B6) Ap 76 x

M58152 (P863) Pl 77 92 x

NC_001409 Pl 77 92 100 x

D14996 (P-205) Ap 77 95 92 92 x

X99752 (Balton-1) Che 78 88 89 89 88 x

AB326225 (MO-5) Ap 77 88 87 87 89 85 x

AJ24 438 (PBM1) Pl 75 93 91 91 92 88 87 x

FN826784

India15ApRep1 77 94 91 91 96 87 88 92 x

FN826785

India15ApRep 2 76 94 90 90 94 87 88 91 93 x

FN826786

India15ApRep 3 76 93 91 91 93 87 88 92 92 98 x

Resu

lts

201

Table 4.25(B): Percentage sequence identities of complete ACLSV-Replicase middle region at amino acid level (below diagonal) among


Ta

Tao -5 B6 P863 Pl P-205 Bal-1 MO-5 PBM1

Ind15Ap

RepMid


AB326224 (B6) Ap 93 x

M58152 (P863) Pl 94 95 x

NC_001409 Pl 94 95 100 x

D14996 (P-205) Ap 94 97 96 96 x

X99752 (Balton-1) Che 94 95 98 98 96 x

AB326225 (MO-5) Ap 91 95 95 95 95 95 x

AJ24 438 (PBM1) Pl 95 98 97 97 97 97 95 x

India15ApRepMid 91 94 95 95 95 95 93 94 x

Resu

lts

202

Table 4.25(C): Percentage sequence identities of complete ACLSV-Replicase 3’end region at amino acid level (below diagonal) among


Ta

Tao -5 B6 P863 Pl P-205 Bal-1 MO-5 PBM1 Ind11 Ind15 RC-Plp


AB326224 (B6) 86 x

Ap M58152 (P863) Pl 86 94 x

NC_001409 Pl 86 94 100 x

D14996 (P-205) Ap 86 94 94 94 x

X99752 (Balton-1) Che 83 91 91 91 91 x

AB326225 (MO-5) Ap 86 94 94 94 94 97 x

AJ24 438 (PBM1) Pl 88 97 97 97 97 94 97 x

India11ApRep3’ 88 97 97 97 97 94 97 100 x

India15ApRep3’ 68 74 74 74 74 71 74 77 74 x

IndiaApRep3’(RC-Plp) 72 86 80 80 80 77 80 83 82 88 x

Resu

lts

203

Table 4.26: Recombination analysis results for all the ACLSV-Replicase sequences. Indian isolates are in red font. Tentative nucleotide (nt)

position in genome mentioned withinin brackets.

Location

analyzed Daughter Major Parent Minor Parent Breakpoints Detected By

Average

P- Value

5’end

FN826785

(India15ApRep2)

apple

AB326224

(B6)

apple

M58152

(P863)

Plum

559-721

(587-749nt)

RDP 4.526 x10-02


SISCAN 2.051 x10-02

AJ24 438

(PBM-1)

plum

FN826785

(India15ApRep2)

apple

D14996

(P-205)

apple

553-868

(581-896nt)

RDP 4.526 x10-02


SISCAN 2.051 x10-02

NC_001409

Plum

FN826785

(India15ApRep2)

apple

D14996

(P-205)

apple

1451-1582

(1479-1610nt)

RDP 4.755 x10-03



MAXCHI 7.080 x10-04


M58152

(P863)

Plum

AB326225

(MO-5)

apple

FN826785

(India15ApRep2)

apple

1472-1569

(1500-1597nt)

RDP 4.755 x10-03



MAXCHI 7.080 x10-04


NC_001409 Plum

FN826784 (India15ApRep1)

apple

AJ24 438 (PBM-1)

plum

1494-1582 (1522-1610nt)

RDP 4.755 x10-03



MAXCHI 7.080 x10-04


Contd…

Resu

lts

204

Location

analyzed Daughter Major Parent Minor Parent Breakpoints Detected By

Average

P- Value

5’end

NC_001409

Plum

FN826785

(India15ApRep2)

apple

X99752

(Balton-1)

cherry

1492-1552

(1520-1580nt)

RDP 4.755 x10-03



MAXCHI 7.080 x10-04


NC_001409

Plum

FN826784

(India15ApRep1)

apple

FN826785

(India15ApRep2)

apple

1534-1577

(1562-1605nt)

RDP 4.755 x10-03



MAXCHI 7.080 x10-04


mid

AB326225

(MO-5)

apple

AB326225

(MO-5)

apple

EU223295

(TaTao5)

Peach

144-357

(4646-4859nt) BOOTSCAN 5.044x10

-04

3’end

AB326225

(MO-5)

apple

AM888397

IndiaApRep3’

(RC-Plp)

apple

M58152

(P863)

Plum

1-18

(5700-5718nt)

RDP 7.715 x10-04


MAXCHI 9.963 x10-03


Resu

lts

205

Fig 4.28: Distance plot showing recombination at 5’end amplicons of ACLSV-Replicase (1472-1569nt) between FN826785

India15ApRep2 (major parent, apple) and AB326225 (MO-5 plum isolate, minor parent) and formation of daughter isolate NC_001409 (Plum)

using RDP ver. 1.08 (Martin et al., 2005)

Resu

lts

Results

206

4.6 DEVELOPMENT OF DIAGNOSTIC TOOL THROUGH

HETEROLOGOUS EXPRESSION OF COAT PROTEIN

4.6.1 ELISA Based Diagnostics

4.6.1.1 Amplification, Cloning and Transformation of Cloned Fragment in E.

coli

The complete CP gene sequence of Indian11 isolate (AM408891, apple) already

cloned in pGEM-T easy vector was used for designing the primers containing Bam

HI and Hind III sites (viz. forward primer 5’-GGATCC ATGGGGGCAGTGCT-3’

and Reverse primer 5’- AAGCTT TTAAACGCAAAGATCAGTT -3’, respectively)

for amplification. Approximately 600bp DNA fragment containing the complete CP

gene was amplified using the designed expression primers (Fig 4.29 A) and sub-

cloned in pGEM-T Easy vector. The recombinant pGEM-T Easy vector containing

the modified CP gene and expression vector pET-32a (+) (Novagen, USA) were

simultaneously digested with Bam HI and Hind III and electrophoresed on 1%

agarose. The gel eluted double digested pET-32a (+) vector (10ng) and modified CP

(50ng) were ligated in-frame, according to manufacturer instructions using T4-DNA

ligase (2 units) (Fermentas, Lithuania), at 160C for 14-16 hours and transformed by

heat shock into E. coli BL-21 competent cells. All the four clones checked using

routine molecular biology techniques (boiling prep plasmid isolation and restriction

digestion) showed desired insert of 600bp (Fig 4.29 B). Three positive clones were

checked by sequencing for the correct orientation of CP gene (inframe cloning).

Single positive clone with correct inframe cloning was used for the standardization

of conditions for optimal expression of CP gene in vitro.

4.6.1.2 Standardization of Optimal Expression Conditions for ACLSV CP

Different IPTG concentrations (0.5, 1, 1.5 mM), temperature combinations (28, 30,

370C) and time intervals (1, 2, 3, 4 hr) were used to standardize optimal expression

(IPTG concentration and temperature) so that expressed protein should be present in

maximum concentration. Analysis by SDS-PAGE of the total proteins from bacterial

cultures 3hr post induction at 280C with 1mM IPTG, revealed the accumulation of

maximum recombinant protein as inclusion body with expected mobility of ~43kDa

(Fig 4.30 B), which was absent in the protein profile from bacteria prior to induction

and from bacteria transformed with empty pET32 (a) vector (transcribing a 20kDa

Results

207

(A) (B)

Fig 4.29: Lane M shows 1kb ladder (Genei, GE Healthcare, USA) (A) lane 1 and 2

show PCR amplified ACLSV-CP using expression primers. (B) Lane 1, 2, 3, 4 show

double digested (Bam HI and Hind III) recombinant pET32 plasmid with ~600bp of

ACLSV-CP as insert.

Fig 4.30: SDS-PAGE gel photograph showing expression of ACLSV CP as His

fusion protein. (A) Protein profile in supernatants, lane 1: Bacteria at OD 0.5, lane 2:

pET vector without insert 3 hours after induction with 1mM IPTG showing 20 kDa

protein, lane 3: pET vector with insert 3 hours after induction with 1mM IPTG

showing no 20kDa protein. (B) Fusion protein expression in pellets of pET vector

with insert after induction with 1mM IPTG at280C, lane M: Prestained protein

marker, lane 1: after 1 hour, lane 2: after 2 hour, lane 3: after 3hour, lane 4: after 4

hour. All show ~43kDa recombinant protein. (C) Lane1: Purified Recombinant

protein, lane 2: last wash buffer flow through during protein purification. Lane M:

Pre stained protein marker (Fermentas, Lithuania) in (A, B, C), (D) Detection of

purified fusion protein using indigenous antisera against ACLSV CP and anti-

rabbit HRP tagged secondary (20)

antibodies after western blotting.

Results

208

native protein) used as controls (Fig 4.30 A).

4.6.1.3 Purification of Expressed Protein

The expressed protein pellet from 1litre culture was resuspended in Bug Buster HT

Protein Extraction Reagent (Novagen, USA) as per manufacturer’s instruction and

purified using precharged His-Bind Column (Novagen, USA) under native

conditions. Purity and integrity of the purified protein was checked by

electrophoresis on 12% SDS-PAGE gel that revealed one distinct band of ~43 kDa

after staining (Fig 4.30 C) and by western blotting (Fig 4.30 D). The concentration of

the purified protein by UV absorption and Bradford method was found to be

~3mg/ml.

4.6.1.4 Raising of Antiserum

For raising antiserum against ACLSV CP immunizations were performed by priming

2 Angora female rabbits, approximately 5 months old, four times at weekly intervals.

Aliquots of purified ACLSV-CP/His fusion protein (0.6 mg) were emulsified with

complete Freund’s adjuvant (FA) (1:1 v/v) and injected subcutaneously into the hind

legs. After 5 weeks, a booster injection emulsified with incomplete FA (0.6mg

protein) was given and rabbits were bled beginning 1 week later at weekly intervals

for 7 weeks. Antiserum / whole serum (obtained after clotting of the blood) against

the ACLSV CP was collected and stored in aliquots at -200C along with 0.02%

sodium azide.

4.6.1.5 Purification of Antibody (Separation of IgG from Whole Serum)

The IgG was extracted and purified from the whole serum using Protein A antibody

purification kit (Genei, India) as per the manufacturer’s instructions. IgG samples

were stored frozen at -200C in 0.5 ml aliquots. The concentration of IgG in the

purified preparation based on the specific extinction coefficient A280 was 1 mg/ml.

4.6.1.6 Preparation of Antibody-Enzyme Conjugate (Using Alkaline

Phosphatase)

A part of antibodies (1.5ml, 1 mg/ml) were tagged with alkaline phosphatase (1.5

mg) by the method of Avrameas (1869). Activity of conjugate was checked by DAS-

ELISA using antibodies produced in rabbit as coating antibodies at optimum

dilution. The conjugate antibodies reacted well with the known positive and negative

Results

209

Table 4.26: Average (triplicates) absorbance values of ELISA with test and

reference kit. Samples from 6-9 have been obtained by pooling leaves of five plants

per host from the cultivars maintained at IHBT (CSIR), Palampur.

S. No. Sample

Absorbance at 405nm

Indigenous Antiserum Commerci

al

Antiserum

(1:1000)

(1:300

) (1:500) (1:1000)

1. Positive control (Kit) 0.960 0.862 0.795 0.782

2. Negative control (Kit) 0.253 0.240 0.231 0.235

3. Blank 0.168 0.160 0.147 0.156

4. Fusion protein 0.718 0.558 0.468 0.479

5. Peach (positive) 0.521 0.487 0.454 0.349

6. Apple Red Spur (5 plants) 0.301 0.255 0.239 0.243

7. wild Himalayan cherry

(5 plants) 0.580 0.526 0.471 0.485

8. Apricot (5 plants) 0.583 0.531 0.462 0.476

9. Plum (5 plants) 0.579 0.523 0.469 0.487

10. Chenopodium amaranticolor

(inoculated-apple) 0.536 0.498 0.463 0.456

11. Phaseolus vulgaris

(inoculated-apple) 0.534 0.485 0.412 0.418

12. Royal Delicious apple

(India11, HP) (positive) 0.615 0.576 0.489 0.502

13. Chenopodium amaranticolor

(inoculated-peach) 0.563 0.496 0.461 0.476

14. Phaseolus vulgaris

(inoculated-peach) 0.528 0.492 0.457 0.472

15. Gala Mast apple

(India15,Gandarbal) 0.545 0.521 0.458 0.496

16. B 9 (Palampur) 0.398 0.356 0.312 0.378

17. Mishri (Cherry, Srinagar) 0.575 0.494 0.452 0.481

18. Rosa domestica 0.496 0.445 0.403 0.416

Results

210

controls up to 1:300 dilution.

4.6.1.7 Evaluation of Antibodies

Antibodies produced in the rabbit were used as coating antibodies in various

dilutions (1:1000, 1:500, 1:300, 1:100.) in DAS-ELISA along with the conjugate

antibodies procured from Bioreba (USA) to identify the titer of the antibodies.

Antibodies produced in rabbit reacted specifically in DAS-ELISA with known

positive and negative samples. The best ELISA readings were obtained at dilution

1:300 which were less ambiguous and more reliable than those given by commercial

kit at 1:1000 dilution (Table 4.26). Sensitivity and specificity of purified IgG against

the ACLSV-CP were also evaluated by western blotting of fusion proteins using

indigenously raised antiserum. Signals obtained by western blotting also indicated to

the indigenous antiserum being specific to ACLSV (Fig 4.30 D).

4.6.1.8 Field Screening to Check the Activity of the Kit

To check efficacy of the indigenous (test) kit (Fig 4.31), different apple cultivars,

pome and stone fruits along with herbaceous hosts were checked for presence or

absence of ACLSV using DAS-ELISA and DTBIA as described earlier. At the same

time samples were also checked by a standard/commercial (reference) kit Bioreba

(USA). Purified fusion protein and Indian ACLSV positive samples (apples and

peach), herbaceous hosts (Chenopodium amaranticolor and Phaseolous vulgaris)

inoculated with positive apple and peach isolate and few random samples were

screened. Peach isolate and herbaceous host inoculated with it were specifically used

in analysis as the isolate showed variation in CP sequence (87% sequence identity at

amino acid level to other Indian isolates). Negative and positive thresholds for DAS-

ELISA were set at two times the mean of healthy control sample absorbance at

405nm. For reference checking commercial ACLSV positive control and negative

control were used. The test was carried out in triplicates. The antiserum reacted

positively with Indian apple and peach isolates; herbaceous host on which ACLSV

India11 (apple, AM408891) isolate culture was maintained and recombinant fusion

protein, whereas no signal was observed in the healthy sample provided with the

commercial kit. It was observed that in some cases the test works better than the

standard or reference kit thus test kit gave stronger positive signal in DAS-ELISA for

Results

211

Fig. 4.31: Indigenously developed ACLSV DAS-ELISA kit.

Fig 4.32: Direct tissue blot immunoassay (DTBIA) using commercial and

indigenous ACLSV antibodies (1:500). Sample 1-inoculated C. amaranticolor-

peach , 2- inoculated P. vulgaris-apple, 3- peach (positive), 4- recombinant coat

protein, 5- positive control (kit), 6-wild Himalayan cherry, 7-apricot, 8-plum, 9-apple

(positive), 10-blank, 11- inoculated P.vulgaris-peach 12- inoculated C.

amaranticolor-apple,13-negative control, 14-apple (negative).

Results

212

some cultivars infected with ACLSV (Table 4.26). The positive control used from

the Bioreba (USA) kit was detected better by the Indian antiserum. Also the peach

isolate which pointed to the possibility of variation among CP isolates from India,

was also detected more reliably by this antiserum (Table 4.26). The ELISA results

indicated that antiserum prepared in the present study was more sensitive for the

detection of Indian isolates of ACLSV in comparison with standard kit. It was found

that in some cases the test kit works better than the standard kit i.e. test kit gave

strong positive signal in DAS-ELISA for some cultivars infected with ACLSV while

the standard kit gave somewhat lesser reaction. Thus kit prepared in the present study

was more sensitive for the detection of Indian strain of ACLSV in comparison to the

standard kit. For DTBIA same plant extracts were plotted on nitrocellulose

membranes (Biorad, USA) and screened with indigenous ACLSV antibodies and

detected using HRP conjugated anti-rabbit antibodies (Roche, USA). Results were

similar to ELISA readings though at 1:500 dilution (Fig 4.32).

4.6.1.9 Keeping Quality of the Kit (Storage Time and Temperature)

To check its keeping quality, DAS-ELISA was conducted using known positive and

negative samples monthly during 2009-2010. A part of antibodies and enzyme

conjugate were kept in refrigerator (4-10°C), whereas other part was kept at room

temperature (20-35°C). It was observed that the kit works well and gave optimum

results when kept at 4-10°C. Keeping the kit reagents at room temperature degrades

their quality and often gave false results. Therefore, it was recommended to keep the

reagents in a refrigerator with temperature of 4-10°C.

4.6.2 Development of PCR Based Diagnostics

4.6.2.1 Standardization RT-PCR for molecular detection

The primer pair was effective in detecting ACLSV infection. The detection was not

affected by the cultivar, host, season of sample collection, plant part used and Taq

polymerases (Genei, India; Invitrogen, USA; Protoscript® AMV Long Amp

TM Taq

RT-PCR Kit, USA). The desired amplification (~800bp) was obtained at annealing

temperature standardized having a range of 45-480 C.

4.6.2.2 Development of Hybridization based Diagnostics

The sequenced CP probes were very effective in detecting ACLSV infection even in

Results

213

winter season irrespective of the CP isolate used.

4.6.2.3 Standardization of Immuno-Capture (IC) - RT-PCR

In IC-RT-PCR indigenous antibodies trapped the virus particles specifically and its

gene was then amplified in RT-PCR using specific primers designed for complete

coat protein (AM490253 and AM490254) thereby restricting the nonspecific

amplifications in RT-PCR. IC-RT-PCR was performed with leaf tissue extracts

(prepared in general extraction buffer as used in ELISA) of wild Himalayan cherry.

Indigenous antibodies specific for ACLSV, specifically bind ACLSV particles

present in the leaf tissue extracts. An alternative freezing and thawing cycle disrupts

the antibody bound virus particles and releases the viral nucleic acid to the solution,

and serves as the template for the amplification of ACLSV genome. The immuno-

capture RT-PCR conditions were also standardized with 1:1000 dilution of

indigenous antibodies and specifically designed primers described in Table 4.12 for

complete coat protein amplification yielded amplicons ~800bp (Fig 4.46). Of all the

samples giving positive amplification only wild Himalayan cherry isolate was

sequenced and submitted to EMBL database (Acc. No. FN666578, FN666579).

These antibodies combine specificity to the target protein and versatility with regard

to other important serological techniques. Amplicons were obtained from Gala Mast

apple cultivar (Kashmir) and wild Himalayan cherry (Palampur). Healthy C.

amaranticolor was used as negative control. No amplification was obtained in the

negative control (Fig 4.33).

Fig 4.33: Gel photograph showing IC-RT-PCR amplicons of ACLSV-CP from leaf

extracts of wild Himalayan cherry (lane 1), Gala Mast apple from Gandarbal (lane 3),

healthy Chenopodium amaranticolor (lane 2). M is 1kb DNA ladder.

Documents

Results - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/7047/9/09_chapter 4.pdf · Results 122 Fig 4.1 I: (A) Chlorosis and leaf puckering in cv. Red Chief apple seedling in