Asian Solanaceous Round Table 2, APSA, Bangkok...
Transcript of Asian Solanaceous Round Table 2, APSA, Bangkok...
Bikash MandalAdvanced Centre for Plant Virology, Indian Agricultural Research Institute, New Delhi, India
Epidemics,
Transmission,
Characterisation and
Diagnosis of
begomoviruses in
chilli leaf curl in India
Asian Solanaceous Round Table 2, APSA, Bangkok 2017
Chilli leaf curl epidemics
Yavatmal District, Maharashtra, India
January 2016
Epidemics
~ 5000 acres during June to March in Yavatmal District, Maharashtra
Leaf curl appeared 10-15 days post transplanting and spread throughout
the fields by October-November.
Many of the farmers were forced to abandon the crop midway and many
uprooted or sown wheat in chilli field
Epidemics
Epidemics 2014, 2015
Madhya
Pradesh (MP)
Leaf curl: Sporadic during 2013-14. >90% during 2014-15
Farmer’s perception: leaf curl was of less importance five years ago, it
emerged due to introduction of new high yielding cultivars
Chilli is a major crop in
Khargone, Dhar and
Badwani districts
Critical Epidemic factors
Chilli
Cultivars
Virus:
Begomovirus
Vector:
Whitefly
Leaf
curl
• High whitefly population, adult 15/leaf
•Whitefly population builds up after intermittent
rains & drought
•High doses and frequent spray (25-30 sprays) of
pesticides used
•Pesticide cocktail along with and several other
micronutrient mixtures
Whitefly (Bemisia tabaci)
Vector
ChiLCV was detected in 30-100% of the field samples indicating emergence of ChiLCV in Jodhpur-a major chilli growing area in Rajasthan state of India.
Epidemics
Virus-vector relation
No. of
WF/
plant
No. of sympt
plants/No.
inoculated (%)
Days to
sympto
ms
Dot-
blot
test
1 4/6 (66.66) 13-18 +
2 3/5 (80.0) 12-13 +
4 5/6 (83.33) 8-10 +
8 6/6 (100) 7-9 +
16 5/6 (83.33) 8-10 +
32 6/6 (100) 8-10 +
Whitefly
transmission of
ChiLCV-[Nar] from
chilli to chilli
Time
(min)
No. of symp
plants/No.
inocul
Days post
inoculation
Dot-blot
test
AAP IAP AAP IAP AAP
IAP5 0/6 0/6 - - - -
10 0/6 0/5 - - - -
15 0/5 0/5 - - - -
30 0/5 0/6 - - - -
60 0/5 6/6 - 11-18 - +
120 0/5 5/5 - 10-11 - +
180 5/5 Nd 11-13 +
240 5/5 Nd 10-13 +
360 4/4 nd 10-11 +
Single WF/plant: 66.6%
>8/plant/plant:100%
Minimum AAP 180 min
Minimum IAP: 60 min
Transmission
Series Serial days
1 2 3 4 5 6
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Persistence of
ChiLCV-[Nar] in
whitefly
The virus persisted in whitefly up
to 5 days post acquisition under
the experimental conditions
•Persistent circulative mode
of transmission
•Efficient transmitter
Transmission
Family Plant species No. of
sympt /No.
inoculated
plantsa
PDI Major
symptom
sb
Asteraceae Ageratum conyzoides 0/6 - -
Caricaceae Carica papaya 4/5 14-17 DLC
Cucurbitaceae Cucumis sativus var. Poinsette 0/6 -
C. melo 0/6 - -
C. moschata 0/6 - -
Citrullus lanatus 0/8 - -
Lagenaria siceraria var. Pusa Naveen 0/6 - -
Luffa acutangula 0/8 -
Euphorbiaceae Croton bonplandianum 0/6 - -
Leguminosae Vigna radiata var. Pusa Baisakhi 0/6 - -
V. mungo 0/6 - -
V. unguiculata var. Pusa Komal 0/6 - -
Glycine max var. Pusa 16 0/6 - -
Phaselous vulgaris 0/6 - -
Malvaceae Abelmoschus esculentus 0/6 - -
Althea rosea var. Pusa Deepika 0/6 - -
var. Pusa Kristina 0/6 - -
var. Pusa Sweta 0/6 - -
Hibiscus esculentus 0/7 -
Gossypium hirsutum var. F1378 0/10 - -
var. GA 0/10 - -
var. H56 0/10 - -
var. RST 9 0/10 - -
var. H 900 0/10 - -
Sida acuta 0/6 - -
Solanaceae Capsicum annuum
var. Pusa Jwala
var. Hybrid 1
var. Hybrid 2
8/10
7/8
8/8
15-17
15-17
15-17
VC, LC
VC, LC
VC, LC
Solanum lycopersicum
var. Pusa Ruby 7/8 11-19 VC, LC
Nicotiana. benthamiana 5/6 10-21 VC, LC
N. tabacum var. Xanthi 6/6 11-13 VC, LC
Solanum melongena var. Pusa Kranti 0/10 - -
S. tuberosum 0/10 - -
Whitefly
transmission of
ChiLCV-[Nar] from
chilli to different
plant species
Host-range of the virus
was limited to
five species
Carica papaya,
Solanum lycopersicum,
Nicotiana tabacum
N. benthamiana)
out of 25 plant species
tested
Early history of chilli leaf curl
1938: Earliest record in Sri Lanka
1954 : First observed in India by Vasudeva
1957-63: Sri Lanka & India- WF transmitted disease, Tobacco leaf curl virus
Rolling circle amplification
Virus
DNA-A shares 68.8-84.40% sequence identity with the other begomoviruses
in the Indian subcontinent
Chilli leaf curl Sri Lanka betasatellite shares 41.5-73.7% identity with other
beta
recombinant begomovirus potentially
originated from the begomoviruses in
Southern India and Sri Lanka
Chilli leaf curl Sri Lanka virus-a begomovirus
Chilli infecting begomoviruses in the Indian subcontinentBegomovirus species reported from chilli
1. Chilli leaf curl virus Senanayake et al., 2006
2. Chilli leaf curl India virus Kumar et al., 2008
3. Chilli leaf curl Vellanad virus Kumar et al., 2012
4. Chilli leaf curl Kanpur virus Singh et al., 2012
5. Chilli leaf curl Palampur virus Kumar et al., 2011
6. Chilli leaf curl Sri Lanka virus Senanayke et al., 2013
7. Pepper leaf curl virus
Begomovirus species originally reported from other host but
naturally infecting chilli
8. Tomato leaf curl Joydebpur virus Shih et al., 2006
9. Tomato leaf curl New Delhi virus Hussain 2000; Khan 2005 KR779820 ChLCV
KT948070 ChLCIV
HM007121 ChLCVV
JN555601 ChLCSV
HM007106 ChLCKV
HM007117 ToLCJoV
XXXXXXXX ChLCV
AF134484 PLCV
KU196750 ToLCNDV
KP313758 MYMIV
KU950430 MYMIV
100
100
76
99
66
100
75
100
Diverse begomoviruses
affect chilli production in
the Indian subcontinent
Partial tandem
dimeric construct
Infectious clone of ChiLCV-Maharashtra
1 2 3 4 M P N
Agroinfection with DNA-A alone
Will be utilised to develop agrobacterium based
screening method for resistance in chilli genotypes
BamHIEcoRI
pCambia2300
BamHI
1.2 kb
2.7 kb
M 1 M 2
1.2 kb2.7 kb
Virus
Diagnosis of begomovirus
Diagnostic methods
ELISA: coat protein highly conserved, PAb ELISA
generally cross react among the species; MAb: rare
PCR: Sequence difference between the species is
randomly distributed over the genome and difficult to
design species specific primers
•The genus Begomovirus : most prolific of all the genera of
plant virus , 322 species
•Multiple begomovirus species infect single crop
•Begomovirus species demarcation criteria: complete genome
sequence (DNA-A) is required
Designing primer through PRIMER BLAST Online tool
To
LC
ND
V
To
LC
Pa
lV
To
LC
Pa
lV
ToLCNDV Clone
pri
me
rs
To
LC
Jo
V
To
LC
ND
V
To
LC
ND
V
To
LC
Jo
V
To
LC
Jo
V
To
LC
Pa
lV
ToLCPalV Clone ToLCJoV Clones
M 1 2 3 M 1 2 3 M 1 2 3
(a) (b) (c)
750bp
PCR with these primers cross-react No virus specific amplification
Diagnosis
New Strategy: Designing of primers with polymorphic 3´ endToLCNDV : species specific primer
(a) Alignment between the species
(b)Alignment within the species 520 530 540 550 1210 1220 1230
.|....|....|....|....|....|....|....| ....|....|....|....|....|....|
ToLCNDV FJ468356.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KF571461.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KF537780.1 GGCCCTTGCAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV HM345979.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KJ000564.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV DQ629101.2 GGTCCTTGTAAGGTGCAGTCGTTCGATGCTAAGAACG-------------------------TTAAGGTACTTAAACACCTGTGTTTTAAAT
ToLCNDV DQ629102.1 GGCCCTTGTAAGGTGCAGTCGTTTGAGTCCAGACATG-------------------------TTTAGGTACTTAAGCACCTGGGTCTTAAAT
ToLCNDV KF551592.1 GGCCCTTGTAAGGTGCACTCGATTGAATCCAGGCTTG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KF551590.1 GGCCCTTGTAAGGGGCAGTCCTTTGAATCTAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KF551589.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCTAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KF551587.1 GGCCCTTGTAAGGTGCACTCCTTTGAATCTAGGCACG-------------------------TCCAGGTACCTAAGGACCTGGGTTTTGAAG
ToLCNDV KF551582.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCTAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTCTGAAA
ToLCNDV KF551580.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KF551577.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV KF551576.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCCAGGCACG-------------------------TCCAGGTACCTAAGGACCTGGGTTTTGAAG
ToLCNDV GQ865546.1 GACCCTTGTAAGGTGCAGTCCTTTGAATCGAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
ToLCNDV JQ897969.1 GGTCCTTGTAAGGTGCAGTCCTTCGAGTCCAGACACG-------------------------TTGAGGTACTTGAGGACCTGGGTTTTGAAT
ToLCNDV HM159454.1 GGCCCTTGTAAGGTGCAGTCCTTTGAATCTAGGCACG-------------------------TCCAGGTACTTAAGGACCTGGGTTTTGAAG
Clustal Consensus * ****************** * ** * * * * ********************* * **
530 540 550 1210 1220 1230 1240
....|....|....|....|....|....| |....|....|....|....|....|....|....|
ToLCNDV KF551589.1 TGTGAAGGCCCTTGTAAGGTGCAGTCCTTT-------------------------- CTATCCAGGTACTTAAGGACCTGGGTTTTGAAGACT
ToLCBV GU474418.1 TGTGAGGGTCCATGTAAGGTCCAGTCATTT-------------------------- TTATTTAAATACTTAAGGACTTGGGTCTTAAAGACC
ToLCPV AM884015.2 TGTGAAGGCCCATGTAAGGTGCAATCTTTT-------------------------- ATATCGAAGTACTTAAGGACTTGGGTTTTGAAAACT
ToLCGV GQ994098.1 TGTGAGGGTCCGTGTAAGGTCCAGTCGTTC-------------------------- TTATTAAGATACTTAAGGACTTGGGTTTTAAATACC
ToLCJo KF551583.1 TGTGAAGGGCCGTGTAAGGTCCAGTCTTTT-------------------------- TTATTTAAATACTTAAGGACTTGGGTCCTAAATACC
ToLCKV HM803118.1 TGTGAAGGCCCATGTAAGGTCCAGTCGTTT-------------------------- TTATTTAAATACTTAAGGACTTGGGTCTTAAATACC
ToLCKeV KF551575.1 TGTGAAGGCCCATGTAAGGTACAATCATTT-------------------------- TTATTTAAATACTTAAGGACTTGGGTCTTAAAGACC
ToLCPaV EU862323.1 TGCGAAGGTCCGTGTAAGGTCCAGTCTTAT-------------------------- CTATTTAAATACTTAAGGACTTGGGATTTAAATACT
ToLCPuV AY754814.1 TGTGAGGGTCCATGTAAGGTCCAGTCTTTT-------------------------- TTATGTAAATACTTAAGGACTTGGGTCTTAAAGACC
ToLCRnV JN676053.1 TGTGAAGGCCCATGTAAGGTGCAGTCTTTT-------------------------- TTATCTAAATACTTAAGGACTTGGGTCTTAAAGACC
ToLCRV DQ339117.1 TGCGAAGGCCCATGTAAGGTCCAGTCGTTT-------------------------- CTATCCAAGTACTTAAGGACTTGGGTCTTAAAGACT
AEV KC795968.1 TGTGAAGGCCCATGTAAGGTCCAGTCGTTT-------------------------- TTATTTAAATACTTAAGGACTTGGGTCTTAAAGACT
CLCBV HM461863.1 TGTGAAGGTCCATGTAAGGTTCAGTCGTTT-------------------------- TTATTTAAATACTTAAGGACTTGGGTCCTAAAGACC
TbCSV JN387045.1 TGTGAAGGCCCATGTAAGGTTCAGTCGTTT-------------------------- TTATTTAAATACTTAAGGACTTGGGTCTTAAAGACC
Clustal Consensus ** ** ** ** ******** ** ** * *** * **************** * ** **
Diagnosis
ToLCBV
ToLCJoVToLCGVToLCPV
ToLCNDV
ToLCPaVToLCKeVToLCKV
ToLCRVToLCRnVToLCPuV
TbCSVAEV
CLCBV
500 1000 1500 2000 270025001
CRAV2
AV1
AC4AC3AC1
AC2
CR
B-797
P-798
B-796
P-799
G-800 G-801
N-794 N-795
J-803J-802
U-783 U-784
Location of specific primers on the DNA-A genome
DNA-A
Diagnosis
Optimization of specific detection of begomovirus species by PCR
•Coned DNA-A of respective begomovirus was used as template•Each primer pair gave amplification with their clone only
56˚C 62˚C 56˚C 60˚C
1 2 3 4 5
M M 1 2 3 4 5
To
LC
ND
V To
LC
BV
To
LC
Pa
lV
To
LC
Jo
V
Ne
ga
tiv
e c
on
tro
l
To
LC
ND
V
To
LC
BV
To
LC
Pa
lV
To
LC
BV
Ne
ga
tiv
e c
on
tro
l
To
LC
ND
V
To
LC
BV
To
LC
Pa
lV
To
LC
Jo
V
Ne
ga
tiv
e c
on
tro
l
To
LC
ND
V
To
LC
BV
To
LC
Pa
lV
To
LC
Jo
V
Ne
ga
tiv
e c
on
tro
l
ToLCNDV Primer ToLCBV Primer ToLCPalV Primer ToLCJoV Primer
Clo
ne
s
700bp
(c)
(d)
M 1 2 3 4
5
(b)
M 1 2 3 4 5
(a)M 1 2 3 4
5 M 1 2 3 4
5 (b) (d)
Identification of annealing temperature
Diagnosis
Collection of tomato samples
Validation of specific detection in the field samples
1.New Delhi2. Haryana 3. Uttar Pradesh 4. Rajasthan 5. Andhra Pradesh 6. Karnataka 7. Orissa 8. Assam
Diagnosis
Testing of field samples of tomato
ToLCNDV primer ToLCPalV primer
ToLCGV primer ToLCBV primer
Selected amplicons were sequenced for further confirmation
Diagnosis
1480 1490 1500 2020 2030 2040 2050 2060 2070
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....
ToLCNDV KF551589.1 GATGTACTCCCCTGTGCGTGAATCCGTGAT-------------------------------AAGGCCGCGCAGCGGCACACACAACATTGGACG
ToLCBV GU474418.1 GATGGGTTCCCCTGTGCGTGAATCCATGGT-------------------------------TAGGCCGCGCAGCGGCATAGACAACGTTTTCTG
ToLCPV AM884015.2 GATGTATTCCCCTGTGCGTGAATCCGTGAT-------------------------------AAGGCCGCGCAGCGGAACACCTCAAATTTGACG
ToLCGV GQ994098.1 GATGAGTTCCCCTGTGCGTGAATCCATGGT-------------------------------GAGGCCGCGCAGCGGGACTGCCGACGTTCTCGG
ToLCJoV KF551591.1 GATGGGTTCCCCTGTGCGTGAATCCATAGT-------------------------------ATGGCCGCGCAGCGGCAGACACCACATTCTCGG
ToLCKV HM803118.1 GATGAGTTCCCCTGTGCGTGAATCCATGGT-------------------------------TAGGCCGCGCAGCGGCACTGACGACGTTCTCGG
ToLCKeV KF551575.1 GATGGGTTCCCCTGTGCGTGAATCCATGGT-------------------------------AAGGCCGCGCAGCGGCACTGACGACATTGTCGG
ToLCPaV EU862323.1 GATGAGTTCCCCTGTGCGTGAATCCATGTG-------------------------------AAGGCCGCGCAGCGGCATCCCTTACATTTTCTG
ToLCPuV AY754814.1 AATGGGTTCCCCTGTGCGTAAATCCATTGT-------------------------------ATGGCCGCGCAGCGGCACTGACAACGTTCTCGG
ToLCRnV JN676053.1 GATGACTTCCCCTGTGCGTGAATCCATGAT-------------------------------ACGGCCGCGCAGCGGCACTGACGACATTCTCGG
ToLCRV DQ339117.1 GATGTACTCCCCTGTGCGTGAATCCGTGAT-------------------------------AAGGCCGCGCAGCGGCACACACCACATTTGACG
AEV KC795968.1 GATGAGTTCCCCTGTGCGTGAATCCATGGT-------------------------------GAGGCCGCGCAGCGGCAGCCATGACATTTTCTG
CLCBV HM461863.1 GATGGGTTCCCCTGTGCGTGAATCCATGGT-------------------------------GGGGCCGCGCAGCGGCATCGACAACGTTATCGA
TbCSV JN387045.1 GATGAGTTCCCCTGTGCGTGAATCCATGAT-------------------------------ATGGCCGCGCAGCGGCACTGACGACATTCTCGA
Clustal Consensus *** ************ ***** * ************* * * **
Designing of common primer for generic detection
Optimization and validation
Diagnosis
S.No. States ToLCNDV ToLCPalV ToLCGV ToLCBV ToLCJoV
1 New Delhi + + + - +
2 Haryana + + + - -
3 Rajasthan + - - - -
4 UP + + + - -
5 AP - - + - -
6 Karnataka - - - + -
7 Orissa - - - - -
8 Assam - - - - -
Distribution pattern of the five tomato infecting begomovirus species in 8 states
Diagnosis
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 M 41 42 43 44 45 46 47 48 49 50 M P
P M 1 2 3 4 5 6 7 8 9 10 11 12 13 P M 14 15 16 17 18 19 20 21 22 23 24
453 bp
P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M
M
P M 15 16 17 18 19 20 21 22 23
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 M P 41 42 43 44 45 46 47 484950 M P
702 bp
ChiLCV ToLCNDV
Specific detection of ChiLCV and ToCNDV in chilli leaf curl in IARI field, Delhi
Out of 50 chilli samples, 44 were positive for ChiLCV and 10 for ToLCNDV
Similarly, ChiLCV specific PCR was developed used to assess
the natural occurrence of ChiLCV and ToLCNDV in
chilli in different places
Diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 M P
18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 34 35 M P
36 37 38 39 40 M P N
453 bp
453 bp
453 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M P N
17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 34 M P N
35 36 37 38 39 40 M P N
702 bp
702 bp
702 bp
ChiLCV ToLCNDV
Out of 40 chilli samples, 31 were positive for ChiLCV , 2 for ToLCNDV & 2 samples for both
Detection of ChiLCV and ToCNDV in chilli leaf curl in Indore, MP
ChiLCV is more predominant begomovirus in chilli
Diagnosis
Distribution
pattern of
begomoviruses
Targeted
breeding
programme
Practical Utility
Species specific PCR diagnostic method for begomoviruses
Diagnosis
BS-35 - (C. fruitiscense x C. chinensis) GKC-29 - (C. annum x C. fruitiscense )Bhut Jolokia - (C. fruitiscense x C. chinensis)
(Kumar et al.,2011)
Against Pepper leaf curl virus
CHUH-4 (C. annum)Pepper Line Nos. 59 & 86 (C. fruitiscense)
(Mondal et al., 2013)
Against leaf curl viruses?
Resistance in chilli
Genetics of resistance
1. PBC-535 × BS-35(highly susceptible) (highly resistant)
F1 Susceptible
2. PBC-535 × Bhut Jolokia(HS) (R)
F1 Susceptible
F2 44S: 12R
Approx 3S:1R ratio
This ratio shows the resistance is governed by monogenic recessive gene
Prakash et al., 2014
Breeding for resistance in chilli
Natural infection
Field screening
Challenge
inoculation
Resistant
sources
Breeding
programme
Resistant
cultivar
Virus
species
specific
Diagnosis
PCR
Whitefly
inoculation
Agro-
inoculation Virus
species
specific
qPCR
T h a n k s
Acknowledgements
Funding: ICAR Platform project on vaccines and diagnostics
Students/SRF
Jayantha Senanayake
Pradeep Kumar
Damini Jwiswal
Scientist
Dr. Anirban Roy