First report of association of begomovirus in yellow mosaic disease of bur cucumber in India

Abstract

In November- December, 2016–2017 Bur cucumber (Sicyos angulatus) plants samples showing severe symptom of yellowing, mosaic and leaf curling were collected from Horticulture research centre (HRC), Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut. Polymerase chain reaction using coat protein gene specific primer (ToLCVAV1F and ToLCVAV1R) gave an amplification of ~ 750 bp, similar to leaf curl virus reported on different crops and weeds. The gene was sequenced and submitted to GenBank with MF471344. BLAST analysis showed the association of Begomovirus
with 99% nucleotide sequence identity with Tomato leaf curl Palampur virus (ToLCPalV) strain India: UP: Matera: Tomato
[KF157552]. Phylogenetic analysis also supported the relationship of the obtained sequence with other begomoviruses as they
clustered in a separate monophyletic cluster with ToLCPalV-Karnataka, India (KY564204, KY564205), India; ToLCPalV Varanasi, India (FJ931537, GQ225738); ToLCPalV-Matera (KF157552), India and ToLCPalV-Allipur, India (HQ848383). Sequence Identity matrix with other begomovirus sequences was found to be in range between (78 and 99%). To our knowledge, this is the first confirmed evidence of a begomovirus infecting weed bur cucumber (Sicyos angulatus) in India.

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Vol.:(0123456789)
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Indian Phytopathology
https://doi.org/10.1007/s42360-018-00107-4
NEW REPORTS
First report ofassociation ofbegomovirus inyellow mosaic disease
ofbur cucumber inIndia
ShivaniKhanna1· SonaliRana1· JitenderSingh1 · ManishaGoyal1· PankajKumar1· NeetuSingh2· R.P.Pant3·
V.K.Baranwal3
Received: 12 June 2018 / Revised: 26 September 2018 / Accepted: 21 December 2018
© Indian Phytopathological Society 2019
Abstract
In November- December, 2016–2017 Bur cucumber (Sicyos angulatus) plants samples showing severe symptom of yellow-
ing, mosaic and leaf curling were collected from Horticulture research centre (HRC), Sardar Vallabhbhai Patel University
of Agriculture and Technology, Meerut. Polymerase chain reaction using coat protein gene specific primer (ToLCVAV1F
and ToLCVAV1R) gave an amplification of ~ 750bp, similar to leaf curl virus reported on different crops and weeds. The
gene was sequenced and submitted to GenBank with MF471344. BLAST analysis showed the association of Begomovirus
with 99% nucleotide sequence identity with Tomato leaf curl Palampur virus (ToLCPalV) strain India: UP: Matera: Tomato
[KF157552]. Phylogenetic analysis also supported the relationship of the obtained sequence with other begomoviruses as they
clustered in a separate monophyletic cluster with ToLCPalV-Karnataka, India (KY564204, KY564205), India; ToLCPalV-
Varanasi, India (FJ931537, GQ225738); ToLCPalV-Matera (KF157552), India and ToLCPalV-Allipur, India (HQ848383).
Sequence Identity matrix with other begomovirus sequences was found to be in range between (78 and 99%). To our knowl-
edge, this is the first confirmed evidence of a begomovirus infecting weed bur cucumber (Sicyos angulatus) in India.
Keyword Tomato leaf curl Palampur virus· Tomato leaf curl New Delhi virus· Coat protein· Phylogenetic analysis
Bur cucumber (Sicyos angulatus) is a climbing or draping
weed often found in woods, along streams and roads, and
in shady, damp places. Recently, in November- December,
2016–2017, symptoms of severe yellowing and mosaic
including curling (Fig.1) were observed in the Horticulture
research centre (HRC), Sardar Vallabhbhai Patel University
of Agriculture and Technology, Meerut, Uttar Pradesh. To
investigate the association of begomovirus with the dis-
ease, six symptomatic and four asymptomatic bur cucumber
leaves samples were collectedand analsed.
Total DNA was extracted from the leaves of symptomatic
and asymptomaticbur cucumber plants by CTAB method
(Doyle and Doyle 1990) and subjected to PCR using coat
protein gene specific primer (TLCVAV1F-R) (Briddon etal.
2002). PCR amplification was performed in 50 ul reaction
mixtures using 1mM of primer, 200mM each of dNTPs,
0.05 unit/ml Taq DNA polymerase, 1× reaction buffer,
1.5mM MgCl2 and 5 µl DNA template either from symp-
tomatic or non symptomatic (healthy) plants. Samples were
amplified for 30 cycles using a thermocycler (Biometra, Ger-
many). Each cycle consisted of initial denaturation 94°C
(10min), denaturation at 94°C (30s), primer annealing at
58°C (30s), and extension at 67°C (45s), and a final exten-
sion of 10min at 72°C.
In Agarose gel electrophoresis of the PCR product, ampli-
cons of the expected size (~ 750bp) was produced from all
six symptomatic samples, confirming begomovirus infec-
tion. No amplification was obtained from non symptomatic
plants. The amplified DNA was excised and eluted from the
gel using QlAquick gel extraction kit (Qiagen, Germany)
(Fig.2). The purified PCR product was ligated into pDrive
cloning vector (Qiagen). Competent Escherichia coli (strain
* Jitender Singh
jeets80@gmail.com
1 College ofBiotechnology, Sardar Vallabhbhai Patel
University ofAgriculture andTechnology, Meerut250110,
India
2 Department ofBiotechnology, Mewar Institute
ofManagement, Vasundhara, Ghaziabad, India
3 Division ofPlant Pathology, Advance Center forPlant
Virology, ICAR-Indian Agriculture Research Institute,
NewDelhi110012, India

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DH 5α) were transformed by standard molecular biology
procedures (Sambrook and Russell 2001). Recombinant
clones were identified with colony PCR and restriction
endonuclease digestion. The two clones were sequenced
in both the orientations at the automated DNA sequencing
facility, Department of Biochemistry, University of Delhi,
South Campus, New Delhi. The partial coat protein gene
sequence obtained was submitted to GenBank with Acces-
sion No. MF471344.
BLAST analysis showed the association of Tomato
leaf curl Palampur virus with 84–99% nucleotide identity
with other isolates ofTomato leaf curl Palampur virus
(ToLCPalV)-Matera, India (KF157552); ToLCPalV-Var-
anasi, India (FJ931537, GQ225738); ToLCPalV-Karna-
taka, India (KY564204, KY564205); ToLCPalV-Allipur,
India (HQ848383). Phylogenetic analysis was performed
using MEGA 6.0 (Tamura etal. 2013) with other closely
related begomoviruses sequences available in GenBank. In
Neighbor-Joining tree, the coat protein gene of virus inecting
Bur cucumber-Meerut (MF471344) were placed in a sepa-
rate monophyletic cluster with ToLCPalV-Karnataka, India
(KY564204, KY564205), India; ToLCPalV-Varanasi, India
(FJ931537, GQ225738); ToLCPalV-Matera (KF157552),
India and ToLCPalV-Allipur, India (HQ848383) (Fig.3).
Sequence Identity matrix of Bur cucumber-Meerut
(MF471344) depicts that it shares highly identical sequence
with other begomoviruses ranging between (78 and 99%).
Therefore results in the present study confirm that the
sequence belongs to a begomovirus isolate.
Earlier, Tomato leaf curl Palampur virus, bipartite bego-
movirus has been reported from India on tomato (Kumar
etal. 2008), Pumpkin (Namrata etal. 2012), Armenian
cucumber (Raj etal. 2015) and Tomato leaf curl New Delhi
virus on Chilli (Singh etal. 2013). In Iran, begomovirus as
new natural hosts has been reported in crop species- tomato,
cucumber, watermelon, bean, Squash, and two weed spe-
cies,Chenopodiumsp. andHeliotropium europaeum (Hey-
darnejad etal. 2013). The virus in Pakistan is reported on
muskmelon (Malik etal. 2011), Bitter gourd (Ali et al.
2010), bell pepper (Tahir and Haider 2006). The increas-
ing incidence of begomoviruses in different cultivated and
weeds species may signal the emergence of a serious threat
to agricultural production throughout the India because it
serves as reservoir or alternative hosts for begomovirus sur-
vival and spread in the absence of the main crops. Thus,
there was a urgent need for additional information on the
diversity and distribution of begomoviruses weeds, which
may serve as virus reservoirs. To our best knowledge, this
is the first report of a begomovirusassociated with bur
Fig. 1 a Healthy bur cucumber
weed. b Bur cucumber showing
typical symptoms of yellowing
and mosaic like symptoms
Fig. 2 Detection of begomovirus in infected bur cucumber samples
using coat protein specific primers. M molecular marker (1KB lad-
der, Fermentas), Lanes 1–6, ~ 750 bp DNA specific to coat protein
region

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cucumber (Sicyos angulatus) from India. But the sequenc-
ing of the entire DNA A component of the begomovirus
infecting bur cucumber is necessary to identify the virus at
species level.
Acknowledgements The authors acknowledge the Vice Chancellor,
Sardar Vallabhbhai Patel University of Agriculture and Technology,
Meerut-250110, Uttar Pradesh and Bioinformatics facility, Department
of Biotechnology, Government of India, for providing financial support
and the facilities to carry out this research work.
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