COMPLETE NUCLEOTIDE SEQUENCE OF A MONOPARTITE TOMATO LEAF CURL NEW DELHI VIRUS AND BETASATELLITE DNA ASSOCIATED WITH LEAF CURL DISEASE OF TOMATO IN INDIA

Abhinav Kumar 1,2 ⃰, Taruna Gupta 3 , Seema Singh 4 and Neera B. Sarin 1 . 1. School of Life Sciences, Jawaharlal Nehru University, New Mahrauli Road, New Delhi-110067, India 2. Dept. of Biotechnology, School of Sciences, Noida International University, GB Nagar, Greater Noida, Uttar Pradesh, India 3. Centre for research studies, Noida International University, GB Nagar, Greater Noida, Uttar Pradesh, India 4. University of Kashmir Hazratbal, Srinagar, Jammu and Kashmir190006, India ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

A begomovirus was isolated from tomato plants showing typical leaf curl and stunting symptoms in tomato fields near the Delhi, India. Viral genomic components were enriched by rolling-circle amplification; PCR amplified and were cloned and sequenced. The genome organization of this virus was found to be similar to those of Old World monopartite begomovirus, with DNA-A and a betasatellite component. The alphasatellite or DNA-B component was not detected. The complete nucleotide sequence of DNA-A shared highest similarity with Tomato leaf curl New Delhi (ToLCNDV) virus while betasatellite DNA showed similarity with betasatellite DNA reported from infected leaves of Tomato, Sunflower and Potato. The data present here strongly support the association of a monopartite ToLCNDV and betasatellite DNA with the leaf curl disease of tomato. In Indian subcontinent, ToLCV is a major problem in tomato-growing regions and several reports on new strains have been documented from New Delhi, Lucknow, Bangalore, Varanasi, Mirzapur, Vadodara, and Western Uttar Pradesh (Chakraborty et al., 2003). In recent years, another component designated as betasatellite (single stranded DNA) has been found to be associated with Old World monopartite begomoviruses. This small, circular DNA has ISSN: 2320-5407

Int. J. Adv. Res. 5(1), 1683-1686
1684 size approximately half (~1.3 kb) to that of DNA-A, and is capable of inducing symptom in plants. It is dependent on DNA-A for replication, encapsidation and transmission (Briddon et al., 2001;Briddon et al., 2008). In addition to betasatellites, monopartite viruses are sometimes found to be associated with another type of satellite designated as alphasatellite, which encodes one replication initiation protein. The role of these alphasatellites in pathogenesis is not yet clears (Briddon et al., 2008). Cloning and sequencing:-PCR amplified product was run on agarose gel and visualized in UV illumination. DNA fragments representing CP gene and betasatellite DNA were excised from the gel and purified using HiPura gel extraction kit (Hi Media Pvt.Ltd. India) following manufacturers instruction. The DNA fragments were cloned into pDrive vector (QIAGEN, Germany). The recombinant plasmid was then transformed into Escherichia coli DH5 cells and positive clones containing desired DNA fragments were sequenced using automated sequencer.  coordinates 1260-14). The ORF βC1 is predicted to encode a 119-amino-acid protein.

1685
The phylogenetic analysis of complete sequence of ToLCNDV under study showed its clustering with ToLCNDV has been reported from Pakistan. The betasatellite DNA clustered with betasatellite reported mostly from non-Solanaceae family. This kind of incidence may occur due to high inoculum pressure or high adaptation capability of the host. This report of monopartite ToLCNDV associated with betasatellite DNA may give some insight into the types of Tomato leaf curl virus infecting tomato crops in north India. There were earlier reports of bipartite ToLCV associated with leaf curl disease of tomato in North India, and monopartite ToLCV in South India (Chowda et al., 2005), but due to more and more sample analysis across the country this opinion is no longer valid, as both types are found from infected tomato plants in all parts of the country (Tiwari et al., 2013). However, monopartite Tomato leaf curl Gujarat virus (ToLCGuV) is now a dominant monopartite begomovirus with betasatellite associated with tomato leaf curl disease in India (Jyothsna et al., 2013).
The search on the available literature and sequences present in NCBI suggests only bipartite ToLCNDV without betasatellite DNA is associated with leaf curl disease of tomato. The report of a monopartite ToLCNDV naturally infecting tomato will add to the variability of ToLCV. Additionally, the data present here strongly supports the association of a monopartite ToLCNDV and non-solanaceous betasatellite with leaf curl disease of tomato in North India.
Under study Fig. 1:-Phylogenetic analysis of DNA-A molecules of this study with other DNA-A molecules of different ToLCV. The optimal tree with the sum of branch length = 0.44812555 is shown. The tree was generated using the Neighbor-Joining method in MEGA 6.0 (software). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Fig. 2:-Dendrogram showing a relationship of betasatellite (this study) with other betasatellite associated with begomoviruses. The optimal tree with the sum of branch length= 0.86863634 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the 1686 branches. The evolutionary distances were computed using the Maximum Composite Likelihood method, and the evolutionary history was inferred using Neighbor-Joining method in MEGA 6.0 software.