Indian Phytopath.

69 (4s)
Phytopathology 69: (4s)
61-64: (2016)
61-64 (2016) 61

RESEARCH ARTICLE

Epidemiology and Diagnosis of Chilli leaf curl virus in

Central India, a major chilli growing region

RAKESH KUMAR, VINAI KUMAR, SREENU KADIRI AND SAIRAM REDDY PALICHERLA*
Division of Biotechnology, J.K. Agri Genetics Ltd, Hyderabad 500 016, Telangana, India

ABSTRACT: Incidence of chilli leaf curl disease on chilli crop in major chilli growing regions of Madhya Pradesh was
surveyed during kharif 2014. The survey revealed that the chilli crop was severely infected (88-100%) by chill leaf curl
virus (ChiLCuV). Amplification of β satellite DNA indicated that the pathogen is a monopartite begomovirus. Full
length sequences of Coat Protein gene and β satellite DNA of the infected samples confirmed that the pathogen
is ChiLCV. Pathogenicity studies demonstrated that these isolates are indeed highly virulent. A new universal
primer to amplify β satellite DNA was also reported in the current study.

Key words: Begomoviruses. Chilli leaf curl virus. Chilli. â Satellite

Chilli (genus Capsicum, family Solanaceae) is an chilli growing region, located in central India, no detailed
important commodity used as a vegetable, spice, epidemiology report was published till date from any
medicinal herb and ornamental plant across the world. other parts of the country. In the current study, an
Chilli is mainly grown in Asian and African countries as extensive survey of the disease incidence was
a cash crop. Asian countries are contributing about 70% conducted in Madhya Pradesh and characterized the
of global dry chilli production with an India’s share of virus. The current study also reports a new set of
about 40% (FAO, 2013). In India, although chilli is grown universal primers, which can be used for diagnosis of a
in most of the states, Andhra Pradesh is the largest broad spectrum leaf curl diseases of various crops
producer (26%) followed by Maharastra (15%), caused by begomoviruses.
Karnataka (11%), Orissa (11%) and Madhya Pradesh
(7%) (Spice Board India, 2012). MATERIALS AND METHODS
In the last two decades, Begomoviruses, largely A roving survey was conducted in 2 major chilli growing
leaf curl viruses have emerged as major threat to districts of Madhya Pradesh, India. To cover maximum
vegetable crops, including chilli causing upto 90% yield area (over 100 km radius), survey was conducted
loss in India (Suresh et al., 2019). Chilli leaf curl virus randomly in five villages of each district viz.,
was first reported in India in 1954, which was later Bhikangaon, Satajana, Bargawana, Dhulwara and
reported in frequent intervals. However, after 2005 the Barsalay of Khargone district; Simlawada, Umar Thana,
virus complex has been emerging rapidly across India Chhatri, Palduna and Nayapura of Ratlam district during
and subcontinent (Kenyon et al., 2014). ChiLCV is November 2014. From each field 50 random plants were
mainly transmitted by whitefly and grafting (Senanayke selected to study symptomology. The average disease
et al., 2012) and diagnosed by simple polymerase chain incidence per field was expressed in percentage. Young
reaction (PCR) or rolling circle amplification (RCA). leaves of clear symptomatic plants with upward leaf
curling, vein clearing and veinal thicking were sampled.
The genomic organization of the virus was first
characterized only in 2007 (Senanayake et al., 2012). In the current study, three new primers were
The virus is mostly monopartite but few reported to have designed for diseased diagnosis and confirmation.
bipartite genome components, DNA A and DNA B, the These primers (CLA7F, CLA5F and CLA8R) are specific
size ranging from 2600 to 2700 nt (Senanayake et al., to chilli leaf curl virus coat protein gene, designed based
2012; Sakata et al., 2008). Monopartite virus consists of on highly conserved DNA A region of 16 ChiLCuV
alpha satellite and β satellite DNA, each of about 1300 whole genome sequence published in NCBI. The current
nt in size (Senanayake et al., 2012; Kumar et al., 2015). study also aimed at designing a new set of primers on â
DNA A genome of monopartite ChiLCuV contains six satellite DNA to detect monopartite virus. The new set of
open reading frames (ORF) (AC1, AC2, AC3, AC4, AV1 primers (CLB36F and CLB37R) were designed using
and AV2) and β DNA contains one ORF to produce conserved 3’ region of 67 begomovirus β satellite DNA
BC1protein (Senanayeke et al., 2012; Bridon et al., sequences published in NCBI (Manuscript under
2002). preparation).
Although the preliminary information of virus DNA was isolated from 100 mg of leaf tissue
incidence was reported from Madhya Pradesh, a major samples collected from various chilli growing regions
*Corresponding author: [email protected] of central India, using CTAB method. DNA was amplified
62 Indian Phytopathology 69 (4s) : 61-64 (2016)

in a PCR reaction. The PCR mixture consists of 1µl of

plant DNA, 2 µl of 2mM dNTP, 1 µl forward and reverse
primers, 2 µl of 10x PCR buffer, 0.2 µl of Taq polymerase
and autoclaved double distilled water, was incubated
in thermocycler (EPGradientS, Eppendorf) with the
following programme: 94°C for 4 min., followed by 35
cycles – denaturation at 94°C for 1 min., annealing
temperature as indicated in Table 1 for 1 min., and
extension at 72°C for 2 min. The reaction was concluded
with a final extension step at 72°C for 10 min. The PCR
product was resolved in 1% agarose gel.

Symptomatic plants of two locations from each Fig. 1. Epidemiology of chilli leaf curl virus infected chilli fields
district were selected and amplified DNA A with specific in Madhya Pradesh, India
primers CLA5F and CLA8R for full length cloning of
coat protein region. The amplicon was eluted from was no significant difference noticed among those
agarose gel and ligated to pTZ57R/T vector (Thermo hybrids in terms of their tolerance to ChiLCV. Disease
scientific, UE) as per manufacturer instructions. The incidence was found to be 100% in Khargone district,
ligated amplicon was transformed into E. coli DH5α but ranged from 88-100% in Ratlam district (Fig. 1, Table
using freeze-thaw method. The β satellite DNA of 2). Due to severe disease incidence, farmers could
Khargone samples were amplified with β settalite DNA collect only 1-2 pickings of their produce, as against
specific primers CLB36F and CLB37R and also cloned more than 5 pickings in case of healthy crop, attributing
into pTZ57R/T. The cloned (pTZ57R) amplicons were to heavy yield losses. As an exception in Bhikangaon
sequenced in ABI 3130 Genetic Analyzer (Applied village, although the disease incidence was noticed as
Biosystems, USA). Sequence alignment and similarity 100%, due to late attack of the disease, the yield loss
matrix were performed using ClustalW algorithm, was found to be limited to last couple of pickings.
BioEdit software and phylogenic tree constructed using
Neighbor Joining method, which is part of Mega 4.0 The sequence alignment result of full length coat
software. protein gene (DNA A) amplified from infected samples
in both the districts confirmed that the pathogen is
To study pathogenicity of the viral strains collected indeed ChiLCV. It is also noticed about 93% similarity
from Khargone and Ratlam, healthy whiteflies were between Coat Protein gene sequence of samples from
released on to the infected chilli branches, freshly Khargoan and Ratlam. Further analysis of the samples
collected and transported overnight to the lab by with β satellite DNA specific primers indicated that these
covering the branch with sterile poly propylene bag viral strains could be monopartite in nature (Fig. 3c).
containing wet tissue papers to maintain humidity. After
24 hrs, whiteflies were collected from infected branches It was also noticed that both Khargone and Ratlam
and released on healthy growing chilli (Byadgi) and ChiLCV isolates segregated along with reference
Nicotiana tabaccum (Xanthi) plants, five plants each, at sequences reported from various parts of India and
the rate of 10-15 flies per plant and kept for overnight in Indian subcontinent in phylogenic dendrogram
a greenhouse at 28oC and 80% humidity. After 12hrs, generated with full length nucleotide sequence of CP
insecticide was sprayed to kill virulent whiteflies and (Fig. 2a) and full length sequence of  DNA (Fig. 2b).
data was collected after 30 days of post inoculation. CP gene of Khargoan isolate shared maximum identity
(~99.4%) with ChiLCV Ahmadabad isolate (JN663846).
Although, Ratlam isolate also showed maximum identity
RESULTS
with Ahmadabad isolate, but it was only up to 93.7%.
Majority of ten villages of two districts surveyed in the The phylogenic dendrogram of  satellite DNA of
current study were covered with commercial hybrids Khargoan isolate showed co-segregation along with
from several hybrid seed companies. However, there ToLCV Rajasthan isolate (AY438558), with ~89.3%


Indian Phytopathology 69 (4s) : 61-64 (2016) 63

identity. However, BC1 gene sequence of ToLCV

Rajasthan isolate was 98.9% identical to Khargoan
isolate.

Artificial disease transmission experiment with two

isolates collected from Khagoan and areas showed
100% infestation in N.tabaccum. However, while the
disease is transmitted to chilli, Khargoan isolate gave
100% infection, but Ratlam isolate showed up to 60%
infection. In either case, it is confirmed that the isolates
collected from both the locations were proved to be
virulent. The symptoms appeared initially as veinal
clearing and later severe leaf curling, followed by
stunting of the plant and no fruit setting due to flower
drop.

DISCUSSION

Chilli leaf curl disease is an important constraint infecting

chilli crops and more prevalent after 2005 across India
(Kumar et al., 2015). However information on disease
incidence and crop loss due to ChiLCV was not reported
from MP. In MP, ChiLCV observed in Jabalpur in 2008
(Kumar et al., 2015). In our study, an epidemic of chilli
Fig. 2a. Neighbor-joining tree of ChiLCV based on Coat Pro- leaf curl disease was observed with up to 100% disease
tein nucleotide sequences
incidence during 2014 in Ratlam and Khargone districts
which is the major chilli growing area in MP. The same
up to 100% disease incidence reported in 2004 in
Rajsthan. The transmission results together with the field
observation of high incidence and severe disease
symptoms indicated the association of a highly virulent
isolates of the virus in the epidemic of leaf curl disease
in Khargone and Ratlam.

CP and beta satellite based molecular

characterization of the virus isolates from epidemic field
revealed the association of ChiLCV with the disease.
The CP gene of both Khargone and Ratlam isolates
are matching with recently reported ChiLCV
Ahmadabad isolate (Bhatt et al., 2016). Current study
noticed that the Khargone isolate did not amplify with
previously reported betasettalite specific primer beta01
and beta02 (Briddon et al., 2003) but could amplify with
Fig. 2b. Neighbor-joining tree of ChiLCV based on β satellite betasatellite begomovirus universal primer CLB36F and
DNA sequences CLB37R, which are reported in the current study.
64 Indian Phytopathology 69 (4s) : 61-64 (2016)

Fig. 3. Molecular detection of chilli infected samples collected from Khargone and Ratlam district of Madhya Pradesh using PCR. (a)
CLA 7F and 8R primers (b) CLA 5F and an 8R primers (c) CLB 36F and CLB37R primers. Lanes showing the PCR amplification of
different villages chilli samples: 1.Sathijana 2. Bargaua 3. Bhikangaon 4. Sonwara 5. Barsalia 6. Shimla buda 7. Umanthana 8. Chhatri
9. Namli 10. Palduna

Betasatellite of Khargone isolate was matching new recom binant begom ovirus and associated
closely with chilli leaf curl Ahmedabad betasetallite and betasatellite DNA infecting Capsicum annuum in India.
Arch. Virol. 161:1389-1394.
falling in India leaf curl virus cluster (Fig. 2b) and
separated from Pakistan betasatelite cluster Briddon, R.W., Bull S.E., Mansoor, S., Amin, I. and
(Senanayake et al., 2012). Markham, P.G. (2002). Universal primers for the PCR-
mediated amplification of DNA-â: a molecule associated
CONCLUSION with some monopartite begomoviruses. Mol. Biotechnol.
20: 315-318.
This is the current epidemiology survey of chilli leaf
curl disease conducted in Madhya Pradesh, a major Kumar R.V., Singh A.K., Singh A.K., Yadav, T., Basu, S.,
Kushwaha, N., Chattopadhyay, B. and Chakraborty,
chilli growing region in central India, which showed S. (2015). Complexity of begomovirus and betasatellite
severe disease prevalence ranging from 88 to 100% populations associated with chilli leaf curl disease in
across the fields, causing severe yield losses. A similar India. J. Gen. Virol. 96:3143–3158
field survey conducted during Kharif 2015 in Khargoan
area (data not published) showed same intensity of the Kenyon, L., Kumar, S., Tsai, W.S. and Hughes, J.A. (2014).
disease, alerting the breeding community to act quickly Virus Diseases of Peppers (Capsicum spp.) and Their
Control. Adv. Virus Res. 90: 297.
to develop ChiLCV resistant varieties for the
sustainability of chilli cultivation in central India. Full Sakata, J., Shibuya. Y., Sharma, P. and Ikegami, M. (2008).
length sequence of CP gene and β DNA of Ratlam and Strains of a new bipartite begomovirus, pepper yellow
Khargone isolates showed similarity to ChiLCV leaf curl Indonesia virus, in leafcurl-diseased tomato and
Ahmadabad isolate, 93.7% and 95% respectively. A yellow-vein-diseased ageratum in Indonesia. Arch. Virol.
new broad spectrum β satellite primers (CLB 36F and 153: 2307-2313.
CLB 37R) reported in the current study might be
Senanayake, D.M.J.B., Varma, A. and Mandal, B. (2012)
important for detection of begomovirus in various Virus-vector relationships, host range, detection and
vegetable crops. sequence comparison of chilli leaf curl virus associated
with an epidemic of leaf curl disease of chilli in Jodhpur,
ACKNOWLEDGEMENTS India. J. Phytopathol. 160:146-155.

Authors extend their gratitude to S.K. Gupta (President, Suresh, L.M., Malathi, V.G. and Shivanna (2013). Molecular
detection of begomoviruses associated with a new
JK Agri Genetics Ltd, Hyderabad, India) for providing
yellow leaf crum kple disease of Cucum ber in
funds and necessary facilities to conduct the current Maharashtra, India, Phytopathology. 66(3): 294-301.
study.
Sonanayake, D.M., Joyasinghe, J.E., Shilpi, Sa Wasala,
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