Asian Solanaceous Round Table 2, APSA, Bangkok 2017

Epidemics, Transmission, Characterisation and Diagnosis of in chilli leaf curl in India

Bikash Mandal Advanced Centre for Plant Virology, Indian Agricultural Research Institute, New Delhi, India Epidemics

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 2014, 2015 Madhya Pradesh (MP)

Chilli is a major crop in Khargone, Dhar and Badwani districts

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 Critical Epidemic factors

Leaf curl

Chilli Cultivars

Vector: : Vector

• 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) Epidemics

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. Virus-vector relation Transmission No. of No. of sympt Days to Dot- WF/ plants/No. sympto blot Whitefly plant inoculated (%) ms test transmission of 1 4/6 (66.66) 13-18 + 2 3/5 (80.0) 12-13 + ChiLCV-[Nar] from 4 5/6 (83.33) 8-10 + 8 6/6 (100) 7-9 + chilli to chilli 16 5/6 (83.33) 8-10 + 32 6/6 (100) 8-10 +

Time No. of symp Days post Dot-blot (min) plants/No. inoculation test inocul AAP IAP AAP IAP AAP IAP Single WF/plant: 66.6% 5 0/6 0/6 - - - - >8/plant/plant:100% 10 0/6 0/5 - - - - 15 0/5 0/5 - - - - 30 0/5 0/6 - - - - Minimum AAP 180 min 60 0/5 6/6 - 11-18 - + 120 0/5 5/5 - 10-11 - + Minimum IAP: 60 min 180 5/5 Nd 11-13 + 240 5/5 Nd 10-13 + 360 4/4 nd 10-11 + Series Serial days Transmission 1 2 3 4 5 6 d 1 d 2 d Persistence of 3 4 d d ChiLCV-[Nar] in 5

d whitefly 6 d 7 d 8 d The virus persisted in whitefly up 9 d 10 to 5 days post acquisition under d 11 d the experimental conditions 12 d 13 d 14 d 15 d •Persistent circulative mode 16 d 17 d 18 of transmission d 19 d 20 •Efficient transmitter d 21 d 22 d 23 d 24 d 25 Family Plant species No. of PDI Major sympt /No. symptom inoculated sb Whitefly plantsa transmission of Asteraceae Ageratum conyzoides 0/6 - - Caricaceae Carica papaya 4/5 14-17 DLC Cucurbitaceae Cucumis sativus var. Poinsette 0/6 - ChiLCV-[Nar] from C. melo 0/6 - - C. moschata 0/6 - - chilli to different Citrullus lanatus 0/8 - - Lagenaria siceraria var. Pusa Naveen 0/6 - - plant species 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 - - of the virus Althea rosea var. Pusa Deepika 0/6 - - Host-range var. Pusa Kristina 0/6 - - was limited to var. Pusa Sweta 0/6 - - Hibiscus esculentus 0/7 - five species Gossypium hirsutum var. F1378 0/10 - - var. GA 0/10 - - var. H56 0/10 - - Carica papaya, var. RST 9 0/10 - - var. H 900 0/10 - - Solanum lycopersicum, Sida acuta 0/6 - - Nicotiana tabacum Solanaceae annuum var. Pusa Jwala 8/10 15-17 VC, LC N. benthamiana) var. Hybrid 1 7/8 15-17 VC, LC var. Hybrid 2 8/8 15-17 VC, LC

Solanum lycopersicum out of 25 plant species var. Pusa Ruby 7/8 11-19 VC, LC tested 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 - - Early history of

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 Chilli leaf curl Sri Lanka virus-a begomovirus

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 infecting begomoviruses in the Indian subcontinent Begomovirus 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. Begomovirus species originally reported from other host but naturally infecting chilli 8. leaf curl Joydebpur virus Shih et al., 2006 9. Tomato leaf curl New Delhi virus Hussain 2000; Khan 2005

100 KR779820 ChLCV

76 KT948070 ChLCIV HM007121 ChLCVV 99 100 JN555601 ChLCSV 66 HM007106 ChLCKV Diverse begomoviruses 100 HM007117 ToLCJoV XXXXXXXX ChLCV affect chilli production in AF134484 PLCV KU196750 ToLCNDV the Indian subcontinent

75 KP313758 MYMIV 100 KU950430 MYMIV Virus Infectious clone of ChiLCV-Maharashtra

BamHI 2.7 kb

1.2 kb

BamHI EcoRI

pCambia2300

M 1 M 2 1 2 3 4 M P N 2.7 kb 1.2 kb

Partial tandem dimeric construct Agroinfection with DNA-A alone

Will be utilised to develop agrobacterium based screening method for resistance in chilli genotypes Diagnosis of begomovirus •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

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 Diagnosis Designing primer through PRIMER BLAST Online tool

ToLCNDV Clone ToLCPalV Clone ToLCJoV Clones

primers

ToLCJoV ToLCJoV ToLCPalV ToLCJoV ToLCNDV ToLCNDV

ToLCPalV ToLCNDV ToLCPalV 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´ end ToLCNDV : species specific primer

(a) Alignment between the species 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 ** ** ** ** ******** ** ** * *** * **************** * ** **

(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 * ****************** * ** * * * * ********************* * ** Diagnosis Location of specific primers on the DNA-A genome

AV2 AC3 AC4 CR AC1 CR DNA-A AV1 AC2 1 500 1000 1500 2000 2500 2700

ToLCNDV ToLCBV ToLCPV ToLCGV ToLCJoV ToLCKV ToLCKeV ToLCPaV ToLCPuV ToLCRnV ToLCRV AEV TbCSV CLCBV G-800 G-801 J-802 J-803

B-796 B-797 U-783 U-784

P-798 P-799 N-794 N-795 Diagnosis Optimization of specific detection of begomovirus species by PCR Identification of annealing temperature

ToLCNDV Primer ToLCBV Primer ToLCPalV Primer ToLCJoV Primer

Clones

ToLCBV

Negative control Negative

ToLCJoV

ToLCBV

ToLCPalV ToLCPalV ToLCPalV

ToLCJoV

ToLCNDV ToLCNDV

ToLCJoV

Negative control Negative control Negative

ToLCBV ToLCBV ToLCBV

ToLCND ToLCNDV V

Negative control control Negative ToLCPalV M 1 2 3 4 5 M 1 2 3 4 1 2 3 4 5 M 1 2 3 4 M 1 2 3 4 5 M M5 1 2 3 4 5 (a) 5 (b) (c) (d)

700bp (b) (d) 56˚C 62˚C 56˚C 60˚C

•Coned DNA-A of respective begomovirus was used as template •Each primer pair gave amplification with their clone only Diagnosis Validation of specific detection in the field samples

Collection of tomato samples

1.New Delhi 2. Haryana 3. Uttar Pradesh 4. Rajasthan 5. Andhra Pradesh 6. Karnataka 7. Orissa 8. Assam Testing of field samples of tomato Diagnosis

ToLCNDV primer ToLCPalV primer

ToLCGV primer ToLCBV primer

Selected amplicons were sequenced for further confirmation Diagnosis Designing of common primer for generic detection

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 *** ************ ***** * ************* * * **

Optimization and validation Diagnosis Distribution pattern of the five tomato infecting begomovirus species in 8 states

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 - - - - - Diagnosis Similarly, ChiLCV specific PCR was developed used to assess the natural occurrence of ChiLCV and ToLCNDV in chilli in different places Specific detection of ChiLCV and ToCNDV in chilli leaf curl in IARI field, Delhi 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 P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M P M 15 16 17 18 19 20 21 22 23 M

453 bp

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 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

702 bp

ChiLCV ToLCNDV Out of 50 chilli samples, 44 were positive for ChiLCV and 10 for ToLCNDV Diagnosis Detection of ChiLCV and ToCNDV in chilli leaf curl in Indore, MP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 M P 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M P N

702 bp 453 bp

17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 34 M P N 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 34 35 M P

702 bp 453 bp

35 36 37 38 39 40 M P N 36 37 38 39 40 M P N

702 bp

453 bp

ChiLCV ToLCNDV

Out of 40 chilli samples, 31 were positive for ChiLCV , 2 for ToLCNDV & 2 samples for both

ChiLCV is more predominant begomovirus in chilli Diagnosis

Species specific PCR diagnostic method for begomoviruses

Practical Utility

Distribution Targeted pattern of breeding begomoviruses programme Resistance in chilli

Against Pepper leaf curl virus 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 leaf curl ? CHUH-4 (C. annum) Pepper Line Nos. 59 & 86 (C. fruitiscense) (Mondal et al., 2013) Genetics of resistance

1. PBC-535 × BS-35 2. PBC-535 × Bhut Jolokia (highly susceptible) (highly resistant) (HS) (R)

F1 Susceptible 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

Agro- inoculation Virus species specific qPCR Natural infection Challenge Resistant Field screening inoculation sources

Breeding Whitefly programme inoculation

Virus species specific Resistant Diagnosis cultivar PCR Acknowledgements

Funding: ICAR Platform project on vaccines and diagnostics Students/SRF Jayantha Senanayake Pradeep Kumar Damini Jwiswal Scientist Dr. Anirban Roy

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