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