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Molecular Characterization of Tomato Leaf Curl Virus (Tolcv) in South Gujarat, India

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Molecular Characterization of Tomato Leaf Curl Virus (Tolcv) in South Gujarat, India Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 473-481 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 473-481 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.603.055 Molecular Characterization of Tomato Leaf Curl Virus (ToLCV) in South Gujarat, India M. Vanthana1*, L. Mahatma1, T.V. Ghevariya1 and R. Saranya2 1Department of Plant Pathology, NMCA, NAU, Navsari, Gujarat 396 450, India 2Department of Plant Pathology, JAU, Junagadh, Gujarat 362 001, India *Corresponding author ABSTRACT The present study was conducted to characterize Leaf Curl Virus infecting tomato in south Gujarat region showing typical leaf curl symptom with upward/downward curling along K e yw or ds with vein clearing of the leaves. A part of DNA-A molecule of ~1200 bp was amplified with a Begomovirus specific primers confirming it to be Begomovirus. On sequencing, a 1125 Begomovirus, bp nucleotide sequence (Accession no- KU921251) was obtained. Amplified fragment had DNA-A, ToLCV, PCR, Phylogeny two genes viz., virus coat protein (V1) gene and pre coat protein (V2) gene. The sequenced virus showed highest identity (94%) with Tomato leaf curl Gujarat virus [Nepal] segment Article Info DNA A, [AY234383.1] followed by 93% sequence identity with thirteen different tomato Begomoviruses which were the strains/isolates of Tomato leaf curl Gujarat virus or Accepted: Tomato leaf curl New Delhi virus [DQ629101.2]. The virus was named as Tomato leaf 10 February 2017 curl Gujarat virus [Nepal] [India:Nvs: LC:Tom:2016] and was abbreviated as ToLCGV Available Online: [Nepal] [India:Nvs: LC:Tom:2016]. Phylogenetic analysis of the sequenced virus with the 10 March 2017 other Begomoviruses of the different crops from the different location indicated that the virus was typically a leaf curl virus. Introduction stranded DNA genomes packaged within Tomato is one of the most important twinned (so-called geminate) particles. The vegetable crops grown throughout the bipartite genome comprises two single- world. It helps in reducing cancer, stranded DNA (DNA-A and DNA-B) cardiovascular diseases and cholesterol components of similar size (2.5-2.8 kb). The (Sachan, 2004) and hence known as nucleotide sequences of DNA-A and DNA-B protective food. The major constraint for are quite different, except for a short tomato growers is the occurrence of Tomato common region of ~200 nucleotides found to leaf curl disease. Among the different be very similar in the two DNAs. Incidence viruses, Tomato leaf curl virus (ToLCV) is of tomato leaf curl disease in India was first the most serious constraints for the reported from northern region (Pruthi and production of tomato in India (Srivastava et Samuel, 1939) and subsequently from al., 1995). ToLCV belongs to genus various parts of the country. The first Begomovirus, family Geminiviridae conclusive etiology of ToLCD in India as a characteristically have circular single- Geminivirus was reported by Muniyappa et 473 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 473-481 al., (1991) and full length sequencing of running on 0.8 per cent agarose gel and ToLCV by Srivastava et al., (1995). In documented the gel by GelDoc (SYNGENE, Gujarat ToLCV was first reported by UK). Chakraborty et al., (2003). Recently, ToLCD has become the prime limiting factor for PCR analysis tomato production in Gujarat. It is persistently transmitted by whitefly Bemisia The virus isolated by Doyle and Doyle (1987) tabaci (Gennadius) (Hemiptera: method was amplified using PCR. A primer Aleyrodidae). The disease causes nearly 40- pair LVF+LVR (LVF=TCTCAACTTCGA 100 per cent yield loss depending upon the CAGCCCATC + LVR=ATAGGTCCAGTG stage of infection and severity (Chakraborty GCGTTTGA) for the amplification of the et al., 2003). A sharp increase in the incidence DNA-A molecule of Begomovirus was used of ToLCD (up to 100%) is being noticed for the amplification of the DNA-A molecule since 1960s after introduction of high of the virus isolated. PCR amplification was yielding tomato varieties. Severe incidences carried out in 25 μl reaction volume of the ToLCD have been observed in all the containing 50 ng genomic DNA, 2.5 μl PCR tomato growing areas of the south Gujarat, buffer (MBI Fermentas, Hanover, USA), 200 however it was difficult to acertain the virus μM dNTPs (Merk, Bangaluru, India), 1.5 U on the basis of symptoms produced. Taq DNA polymerase (MBI Fermentas) and Therefore, attempts were made to characterize 0.4 μM primer using a thermal cycler the virus. (Eppendorf, Germany). Materials and Methods The programme was performed as 1 cycle of 940 C for 2 min and 35 cycles of 940 C for 45 Source of viral sample 0 0 sec, 52 C for 1min, 72 C for 1min then a Samples from the tomato crop sown in the final extension step at 720 C for 5min. The field of Navsari Agricultural University were PCR products were run on 2.0 per cent (w/v) collected showing typical leaf curl symptom agarose gel in 0.5X TBE buffer at 100 mV for with upward/downward curling along with 1 hr. Gels with amplified fragments were vein clearing of the leaves. visualized and photographed under UV light using SYNGENE Bio imaging system. Molecular characterization Purification and sequencing Isolation of viral DNA The amplified product of PCR was purified For DNA extraction, fresh leaf (500mg tissue) and used for sequencing. Sequencing was from the infected plant were taken and done by adding Terminator Ready Reaction homogenized in Eppendorf tube using liquid Mix (Big Dye sequencing kit 3.1v provided nitrogen. After complete homogenization, by Applied Bio systems). 700μl of CTAB extraction buffer was added and incubated the Eppendorf tube at 60-650 C Data were retrieved from the sequencer and for one hour in water bath. Isolation of DNA further analysed for similarity index using was done by the CTAB method given by NCBI-BLASTN and multiple nucleotide (nt) Doyle and Doyle, 1987 with slight sequence alignments using CLUSTALW modification. The quantity of the isolated (2.1). ORFs of the sequence was also obtained DNA was measured using spectrophotometer using Open Reading Frame finder of the (Nanodrop). DNA quality was tested by NCBI software. 474 Int.J.Curr.Microbiol.App.Sci (2017) 6(3): 473-481 Phylogenetic tree analysis Gujarat virus [Nepal] segment DNA A, [AY234383.1] followed by 93% sequence Phylogenetic tree was constructed by using identity with thirteen different Begomoviruses online software Neighbor-joining method of tomato which were strains/isolates of with 1000 bootstrap replication in the MEGA Tomato leaf curl Gujarat virus or Tomato version 4.0 (Tamura et al., 2007). The leaf curl New Delhi virus (Table 1). For the representative species, strains and variants of nomenclature and demarcation of species, different Begomovirus species from the strains and variants of the species of the virus different crops and geographical location the recent criteria proposed by Fauquet et were selected for the phylogenetic analysis. al., (2008) and subsequent guidelines given by ICTV (Anon 2015) were used. Results and Discussion Accordingly the sequenced isolate has been considered as tentative strain of Tomato leaf Symptoms of the disease observed during the curl Gujarat virus [Nepal] and named as investigation were typical leaf curl symptoms Tomato leaf curl Gujarat virus [Nepal] with the upward/downward curling along [India: Nvs:LC:Tom: 2016] and is with the vein clearing of leaves. Fruit, if abbreviated as ToLCGV [Nepal] [India: produced at all, were small, dry and Nvs:LC:Tom: 2016]. Representative species, unmarketable. Leaves were often stiff, strains and variants of different Begomovirus thicker and of leathery texture rather than species from the different crops and flaccid. Affected plants grew slowly and geographical location were selected for the became stunted or dwarfed. The flowers phylogenetic analysis (Table 2). The appear normal. No chlorotic or yellowing of Dendrogram was constructed from the the leaf lamina could be seen. Viral DNA was aligned sequences using the neighbor- successfully isolated from symptomatic joining method and bootstrap option of Tree younger leaves of Tomato plants by CTAB conversion (1000 bootstrap replicates). A method. PCR amplification to amplify a part total of 56 different viruses including recently DNA-A molecule showed ~1200 bp band of sequenced virus during present investigation DNA-A molecule. Amplified fragment was [Accession no. KU921251] were aligned purified and sequenced in an automated together in a phylogenetic tree (Fig. 1). The DNA sequencer by the Cycle sequencing virus under investigation was found aligned method and a 1125 bp nucleotide sequence closely with different strains of Tomato Leaf was obtained. This was deposited to the Curl Gujarat virus. All the Begomovirus Genebank, NCBI and Accession no. claded in two distinct clads. KU921251 was obtained. ORF obtained by the ORF finder of the NCBI software One clad comprised of all the Begomoviruses indicated that the sequence is having two infecting different crops viz., cotton, cassava, ORF. Comparison of the sequence with the okra, gourds, tomato, papaya and chilli. other standard universal ORF of the DNA-A Another clad consisted of species, strains molecule indicated that the amplified and variants of different Begomovirus fragment have two genes viz., virus coat infecting legumes were obtained. This protein (V1) gene and pre coat protein (V2) indicated that the Begomovirus infecting gene.
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