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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
ACLSV ASGV ASPV ApMV PNRSV PPV
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
ACLSV ASGV ASPV ApMV PNRSV PPV
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
ACLSV ASGV ASPV ApMV PNRSV PPV
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
ACLSV ASGV ASPV ApMV PNRSV PPV
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
+ Sign indicates positive results in ELISA and blank spaces indicate negative result
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
+ Sign indicates positive results in ELISA and blank spaces indicate negative result
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
ACLSV ASGV ASPV ApMV PNRSV PPV
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-
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)
16. CAE52469 M76 P Albania (Alb) 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
S. no Accession no. Isolate name CP Country Source
21. CAE52481 M119 P Albania (Alb) apple (Ap)
22. CAE52482 M139 P Albania (Alb) apple (Ap)
23. CAE52483 M62 P Albania (Alb) apple (Ap)
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)
36. AJ586647 PE151 P Lebanon (Leb) peach (Pe)
37. AJ586648 PE152 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
S. no Accession no. Isolate name CP Country Source
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)
57. AJ586629 Apr-110 P Spain (Spa) apricot (Apr)
58. AJ586622 Apr-103 P Spain (Spa) apricot (Apr)
59. AJ586630 Apr-20 P Italy (Ita) apricot (Apr)
60. AJ586632 Apr-60 P Spain (Spa) apricot (Apr)
61. AJ586633 Apr-61 P Spain (Spa) apricot (Apr)
62. AJ586634 Apr-62 P Spain (Spa) 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
AM494505In MAAVLNLQLK VDADLKAFLA AEGRPLHGKT GAILEQTLEA IFANIAIQGT SEQTEFLDVL VEVKSMEDQK VVGSFNLKEV VNLIKIFRTT SSDPNISNMT
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
AM494505In FRQVCEAFAP EARNGLVKLK YKGVFTNLFS TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKSEN EAKMSSVTTD LCI
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).
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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,
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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
AM882705In MAAVLNLQLK VDADLKAFLA AEGRPLHGKT GAILEQTLEA IFANIAIQGT SEQTEFLDVL VEVKSMEDQK VVGSFNLKEV VNLIKIFRTT SSDPNISNMT
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
AM882705In FRQVCEAFAP EARNGLVKLK YKGVFTNLFS TMPEVGGKYP ELMFDFNKGL NMFIMNKAQQ KVITNMNRRL LQTEFAKSEN EAKMSSVTTD LCI
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…
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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)
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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).
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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
lts
174
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
lts
175
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
lts
176
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
lts
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
lts
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)
24. AM709776 Palampur India13 P (3’end) apple (Ap)
25. AM709777 Palampur India14 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
CHIMAERA 3.399 x10-03
Complete
MP
(~1408bp)
FN673831
India15Apmp
apple
EU223295
(TaTao-5)
Peach
AB326225
(MO-5)
apple
6-27
RDP 1.566x10-06
GENECONV 1.067 x10-03
MAXCHI 2.522 x10-02
CHIMAERA 1.316 x10-02
FN673831
India15Apmp apple
EU223295
(TaTao-5) Peach
AB326224 (B6) apple
839-946
RDP 3.178 x10-02
GENECONV 3.926 x10-03
BOOTSCAN 2.534 x10-02
NC_001409
plum
FN673831
India15Apmp
apple
X99752
(Balton-1)
cherry
895-966
RDP 1.824 x10-02
BOOTSCAN 2.681 x10-02
CHIMAERA 7.271x10-03
M58152
(P863) Plum
FN673831
India15Apmp
apple
AB326225
(MO-5)
apple
4-18
RDP 1.566x10-06
GENECONV 1.067 x10-03
MAXCHI 2.522 x10-02
CHIMAERA 1.316 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
BOOTSCAN 1.928 x10-04
CHIMAERA 8.502 x10-03
SISCAN 3.114x10-02
AB326225
(MO-5)
apple
FN673831
India15Ap-1
NC_001409
plum
1732-
1828
RDP 1.259x10-02
GENECONV 1.596x10-02
BOOTSCAN 3.554 x10-03
MAXCHI 7.118 x10-09
CHIMAERA 4.032 x10-03
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
AM882704In 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
AM494505In 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
AM882705In D.KE.D...A E..V...... .......... ........P. ....G..... ..T.H...N. ...RA...EI H..P...GVI ...GG.QFN. ...D...... ..QY.L.P.V
AM931534In 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
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
AM882704In 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
AM494505In 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
AM882705In D.KE.D...A E..V...... .......... ........P. ....G..... ..T.H...N. ...RA...EI H..P...GVI ...GG.QFN. ...D...... ..QY.L.P.V
AM931534In 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
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)
10. FN826785 India (Kashmir) India15ApRep2 5’end apple (Ap)
11. FN826786 India (Kashmir) India15ApRep3 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)
9. FN826784 India (Kashmir) India15ApRep1 5’end apple (Ap)
10. FN826785 India (Kashmir) India15ApRep2 5’end apple (Ap)
11. FN826786 India (Kashmir) India15ApRep3 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)
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)
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(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.
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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
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Table 4.25(B): Percentage sequence identities of complete ACLSV-Replicase middle 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
Ind15Ap
RepMid
EU223295 (TaTao 5) Pe x
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
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Table 4.25(C): Percentage sequence identities of complete ACLSV-Replicase 3’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 Ind11 Ind15 RC-Plp
EU223295 (TaTao 5) Pe x
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
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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
BOOTSCAN 2.461 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
BOOTSCAN 2.461 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
GENECONV 2.930 x10-03
BOOTSCAN 2.165 x10-04
MAXCHI 7.080 x10-04
CHIMAERA 1.490 x10-02
M58152
(P863)
Plum
AB326225
(MO-5)
apple
FN826785
(India15ApRep2)
apple
1472-1569
(1500-1597nt)
RDP 4.755 x10-03
GENECONV 2.930 x10-03
BOOTSCAN 2.165 x10-04
MAXCHI 7.080 x10-04
CHIMAERA 1.490 x10-02
NC_001409 Plum
FN826784 (India15ApRep1)
apple
AJ24 438 (PBM-1)
plum
1494-1582 (1522-1610nt)
RDP 4.755 x10-03
GENECONV 2.930 x10-03
BOOTSCAN 2.165 x10-04
MAXCHI 7.080 x10-04
CHIMAERA 1.490 x10-02
Contd…
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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
GENECONV 2.930 x10-03
BOOTSCAN 2.165 x10-04
MAXCHI 7.080 x10-04
CHIMAERA 1.490 x10-02
NC_001409
Plum
FN826784
(India15ApRep1)
apple
FN826785
(India15ApRep2)
apple
1534-1577
(1562-1605nt)
RDP 4.755 x10-03
GENECONV 2.930 x10-03
BOOTSCAN 2.165 x10-04
MAXCHI 7.080 x10-04
CHIMAERA 1.490 x10-02
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
GENECONV 5.773 x10-03
MAXCHI 9.963 x10-03
CHIMAERA 6.746 x10-03
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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)
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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
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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
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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
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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
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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.