Indian Journal of Biotechnology Vol 7, April 2008, pp 214-217

Molecular characterization of Tobacco streak virus causing soybean necrosis in India

N Arun Kumar1,2, M Lakshmi Narasu2, Usha B Zehr1 and K S Ravi1* 1Mahyco Research Center, Jalna-Aurangabad Road, Dawalwadi, Post Box No. 76, Jalna 413 203, India 2Center for Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 072, India Received 13 March 2007; revised 3 May 2007; accepted 13 September 2007

A virus isolate infecting soybean (Glycine max L.) with characteristic symptoms of necrosis was collected from various locations of Maharashtra, India. The virus was detected as Tobacco streak virus (TSV) by direct antigen-coating-enzyme- linked immunosorbent assay (DAC-ELISA) using TSV specific antiserum. ELISA positive TSV soybean isolates upon mechanical transmission onto indicator hosts, viz. Vigna unguiculata cv. C-152, Glycine max and Nicotiana tabacum cv. Xanthi, produced characteristic local necrotic lesions on primary inoculated leaves, followed by systemic infection. Coat protein (CP) gene from a representative soybean isolate (TSV-SB) was amplified using TSV CP specific primers. The amplicon of ~750 bp was cloned and sequenced. The CP gene consists of 717 nucleotides, potential of coding a polypeptide of 238 amino acid residues. The CP gene of TSV-SB isolate shared 98.3 to 99.3% nucleotide and 96.6 to 98.3% amino acid sequence homology with the TSV isolates characterized from India. With TSV-WC (White clover isolate from America, type strain) and TSV-BR (Soybean isolate from Brazil), TSV-SB isolate shared 88.7% and 79.2% amino acid sequence homology, respectively. Phylogenetic tree constructed based on amino acid sequence analysis showed a close clustering of TSV-SB isolate with other TSV isolates from India than with the American and Brazilian strains. Based on CP gene sequence analysis, it is concluded that the TSV-SB isolate is a strain of TSV that is prevalent in India. This is the first report of molecular characterization of TSV infecting soybean from India.

Keywords: coat protein gene, Tobacco streak virus, soybean

Introduction from USA7,8 and Brazil9,10, where it contributed to A disease characterized as bud blight with yield losses of 25 and 100%, respectively. symptoms of chlorosis, necrosis of leaves, stem, and In recent years, TSV has emerged as an important buds, and stunting was reported to cause serious virus infecting several economically important crops damage to soybean (Glycine max L.) in India1. Based in India4,5,11-19. In India, based on coat protein (CP) on the serological and molecular characterization, the gene sequence analysis, the virus isolate infecting causal agent of soybean bud blight was identified as a sunflower crop was identified as a strain of TSV strain of Groundnut bud necrosis virus (GBNV)2,3. In distinct from the type strain, TSV-WC (White clover recent years, Tobacco streak virus (TSV)—an isolate from America)19-20. Further, the CP gene economically important virus in India—was reported sequence analysis of the TSV isolates characterized to produce similar symptoms of necrosis4,5 as from various crops and locations of India was described in the bud blight disease of soybean3. Such reported to share 99 to 100% nucleotide sequence similarity in the necrotic symptoms due to TSV or homology among each other and shared 88 to 89% GBNV infections was earlier reported on groundnut6. sequence homology with the type strain19-21. Recently, Serological reaction5 and reverse transcription a TSV isolate causing soybean bud blight disease in polymerase chain reaction (RT-PCR)4 confirmed the Brazil (TSV-BR) was reported to be a distinct strain TSV infection in soybean. The necrosis of soybean of TSV, which shared 81.3 and 80.7% nucleotide was reported to be prevalent upto 40% in Maharashtra sequence homology with the CP gene of TSV-WC resulting in severe yield losses5. The association of and TSV-MB (mungbean isolate from India), TSV with bud blight of soybean was earlier reported respectively10. However, the complete molecular ______identity of the soybean isolate causing necrosis * Author for correspondence: Tel: 91-2482-262001; Fax: 91-2482-234621 disease in India was not addressed. Thus, the present E-mail: [email protected] study was aimed to determine the molecular KUMAR et al: CHARACTERIZATION OF TOBACCO STREAK VIRUS SOYBEAN ISOLATE 215

characterization and phylogenetic relationship of the Cloning, Sequencing and Sequence Analysis TSV soybean isolate from India. The amplicon was gel eluted using QIAEXII gel extraction kit (Qiagen Inc., Chatsworth, CA, USA) Materials and Methods and ligated into pGEM-T Easy vector (Promega, Madison, WI, USA) following the manufacturer’s Virus Isolates instructions. The ligated product was transformed into Soybean plants showing symptoms of chlorosis, Escherichia coli DH5α competent cells by following mosaic mottling, necrosis of leaves, petiole, stem and 22 pods were collected from Jalna, Beed and Osmanabad standard molecular biology procedures . regions of Maharashtra. Symptomatic samples Recombinant clones were identified by restriction collected from various locations were diagnosed by digestion analysis using NcoI and XhoI enzymes. The recombinant TSV CP plasmid was sequenced with direct antigen-coating-enzyme-linked immunosorbent universal sequencing primers (T7 and M13 reverse) assay (DAC-ELISA) using TSV specific polyclonal antiserum (Mahyco Research Center)5. The ELISA using BigDye termination cycle sequencing kit positive samples were further inoculated onto Vigna (Applied Biosystems, USA) and analyzed in an unguiculata cv. C-152 (cowpea), G. max and automated DNA sequencer (ABI Prism377, Applied Nicotiana tabacum cv. Xanthi (tobacco) by Biosystems). Nucleotide sequence data was compiled mechanical sap inoculations, using 0.05 M phosphate using ContigExpress and AlignX program in Vector buffer, pH 7.0 containing 0.075% (v/v) mono- NTI version 6.0 software. Translations of the primary thioglycerol, under greenhouse conditions. nucleotide sequences, multiple sequence alignment and the sequence homologies with the corresponding Reverse Transcription-Polymerase Chain Reaction (RT-PCR) CP gene sequence of other TSV isolates were To determine the identity of the virus isolate determined using Bio Edit Sequence Alignment infecting soybean, a representative isolate from Editor. Based on the sequence homologies, a Osmanabad region was analyzed molecularly. Total phylogenetic tree was generated using NTSYS-pc

RNA isolated from the infected and healthy soybean version 2.11 software. The CP gene sequences of TSV leaf tissue, using RNeasy plant extraction kit (Qiagen isolates used in the present sequence analysis were Inc., Chatsworth, CA, USA), was used as a template downloaded from NCBI GenBank (Table 1). in RT-PCR with TSV CP specific primers. The Results and Discussion forward (5′-CATGCCATGGCAATGAATACTTT Infected soybean plants showing the symptoms of GATCCAA-3′) and reverse (5'-CCGCTCGAG chlorosis and necrotic lesions on leaves, veins, mid- ATCTTGATTCACCAGGAAAT-3') primers were rib, petioles, stem and pods reacted positive with the designed to amplify the complete coding region of TSV polyclonal antiserum in DAC-ELISA, TSV CP. The primers were linked to adapters suggesting association of TSV with the necrosis containing NcoI and XhoI restriction sites disease. Of 30 soybean samples collected from Jalna, (underlined) at 5′ end of the forward and reverse Osmanabad and Beed regions of Maharashtra, 25 primer, respectively. Amplification was performed in ® Table 1—NCBI accession numbers of coat protein gene sequence an automated PCR machine (GeneAmp PCR system of Tobacco streak virus isolates characterized from India, USA 9700, PE Applied Biosystems) programmed for one and Brazil cycle of cDNA synthesis at 45°C for 45 min, followed Host Location Isolate code Acc.No. by denaturation of reverse transcriptase at 94°C for 3 Sunflower Tamil Nadu TSV-SF Cbt AF400664 min and amplification of 35 cycles comprising the Sunflower Maharashtra TSV-SF A'bad AY061928 following parameters: 94°C of denaturation for 45 Sunflower Karnataka TSV-SF Blr AY061929 sec, 60°C of annealing for 45 sec, and at 72°C of Sunflower Andhra Pradesh TSV-SF Hyd AY061930 extension for 1 min, followed by a cycle of final Sunflower Tamil Nadu TSV-SF Cbt AU1 DQ233634 extension at 72°C for 10 min. RT-PCR was carried Mungbean Tamil Nadu TSV-MB AF515823 Cotton Maharashtra TSV-CO AF515824 out using one-step Qiagen RT-PCR kit (Qiagen Inc., Sunhemp Tamil Nadu TSV-SH AF515825 Chatsworth, CA, USA). The RT-PCR product was Chilli Uttar Pradesh TSV-CH AY590139 analyzed on a 0.8% agarose gel, stained with ethidium Cowpea Tamil Nadu TSV-CP DQ058079 bromide (0.5 µg/mL) and viewed under Urdbean Tamil Nadu TSV-UB DQ225172 White Clover America TSV-WC X00435 transilluminator. Soybean Brazil TSV-BR AY354406 216 INDIAN J BIOTECHNOL, APRIL 2008

reacted to TSV antiserum with absorbance values at conserved CP amino acid sequence of the Indian TSV A405 nm ranged from 0.48 to 1.80. The symptomatic isolate. With TSV-WC (American) and TSV-BR leaf (ELISA positive) samples upon mechanical sap (Brazilian), CP of TSV-SB isolate shared 88.7 and inoculation produced necrotic lesions on the 79.2% amino acid sequence homology, respectively. inoculated primary leaves of cowpea cv. C-152, The phylogenetic analysis based on the deduced soybean and tobacco followed by systemic necrosis amino acid sequences showed a close clustering of confirming the natural infection of TSV on soybean TSV-SB with other Indian TSV isolates, irrespective plants. of the hosts and the locations from which/where the In RT-PCR analysis, total RNA isolated from the isolates were characterized; while it diverged from the symptomatic soybean leaf tissue amplified ~750 bp type strain as well as the Brazilian soybean isolate fragment corresponding to the CP gene of TSV. No (Fig. 1). such amplification was observed when total RNA In the present study, the association of TSV with extracted from a healthy soybean leaf tissue (data not necrosis disease of soybean in India was established shown). Sequence analysis showed that the amplified based on serology, transmission and CP gene product was 717 nucleotides long coding for a protein sequence analysis. CP gene of TSV-SB isolate shared of 238 amino acids, which was similar to the size of a high degree of sequence homology with other TSV CP gene of the TSV isolates characterized from India17-18,20-21 and Brazil (TSV-BR)10. Whereas, the CP sequence of TSV soybean (TSV-SB, present study) and other Indian isolates possessed an additional amino acid residue at 25th position compared to the 237 amino acid encoding CP of TSV- WC, type strain23. The primary nucleotide sequence of TSV-SB isolate has been deposited in NCBI database (Acc.No. AY940151). Comparative sequence analysis revealed that the CP gene of TSV- SB shared 98.3 to 99.3% and 96.6 to 98.3% homologies at nucleotide and amino acid sequence levels, respectively with the TSV isolates characterized from other crop species and locations of India (Table 2). TSV-SB isolate showed 5 amino acid substitutions in the CP at 7th, 25th, 120th, 154th and 157th positions from glycine to serine, arginine to Fig. 1—Phylogenetic tree showing the relationship of Tobacco streak virus soybean isolate (TSV-SB) with other isolates from serine, phenylalanine to leucine, valine to alanine and India, America (TSV-WC, type strain) and Brazil (TSV-BR) valine to alanine, respectively with reference to the based on the coat protein sequence homologies.

Table 2—Per cent nucleotide (above the diagonal line) and amino acid (below the diagonal line) sequence homologies in the coat protein gene of Tobacco streak virus soybean isolate (TSV-SB) and other TSV isolates characterized from India, USA and Brazil Sl. Isolate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 TSV-SB * 99.3 98.8 99.3 99.1 99.1 99.0 99.3 99.1 98.3 99.1 99.0 88.1 80.3 2 TSV-SF Cbt 97.8 * 99.5 100.0 99.8 99.8 99.7 100.0 99.8 99.0 99.8 99.7 88.7 81.1 3 TSV-SF A'bad 97.4 99.5 * 99.5 99.4 99.4 99.3 99.5 99.4 98.8 99.4 99.3 88.8 80.7 4 TSV-SF Blr 97.8 100.0 99.5 * 99.8 99.8 99.7 100.0 99.8 99.0 99.8 99.7 88.7 81.1 5 TSV-SF Hyd 97.8 100.0 99.5 100.0 * 99.7 99.5 99.8 99.7 98.8 99.7 99.5 88.5 80.7 6 TSV-SF Cbt AU1 97.8 100.0 99.5 100.0 100.0 * 99.5 99.8 99.7 98.8 99.7 99.5 88.7 81.1 7 TSV-MB 97.4 99.5 99.1 99.5 99.5 99.5 * 99.7 99.5 98.7 99.5 99.4 88.4 80.7 8 TSV-CO 97.8 100.0 99.5 100.0 100.0 100.0 99.5 * 99.8 99.0 99.8 99.7 88.7 81.1 9 TSV-SH 97.8 100.0 99.5 100.0 100.0 100.0 99.5 100.0 * 98.8 99.7 99.5 88.5 81.1 10 TSV-CH 96.6 98.7 98.3 98.7 98.7 98.7 98.3 98.7 98.7 * 98.8 98.7 88.2 80.3 11 TSV-CP 97.8 100.0 99.5 100.0 100.0 100.0 99.5 100.0 100.0 98.7 * 99.5 88.8 81.1 12 TSV-UB 97.4 99.5 99.1 99.5 99.5 99.5 99.1 99.5 99.5 98.3 99.5 * 88.5 80.7 13 TSV-WC 88.7 89.9 89.4 89.9 89.9 89.9 89.4 89.9 89.9 89.4 89.9 89.4 * 81.3 14 TSV-BR 79.2 80.7 80.3 80.7 80.7 80.7 80.3 80.7 80.7 79.5 80.7 80.3 82.8 * KUMAR et al: CHARACTERIZATION OF TOBACCO STREAK VIRUS SOYBEAN ISOLATE 217

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