Chapter-4 Discussion Discussion 5.1 Biological Characterization

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2 Withania somnifera (L.) Dual also known as Indian ginseng of the family Solanaceae, is a multipurpose plant of immense therapeutic value. It is an evergreen, erect or spreading, rarely decumbent shrub and exhibits extensive phenotypic and chemical variability. The plant is native to drier parts of India and The Old World. It is used in a variety of Ayurvedic, Homeopathic and Unani medicines (Anon., 1998). The therapeutic value of W. somnifera is ascribed to various bioactive molecules synthesized and accumulated in its roots and leaves. The plant can be compared favourably to the world s most renowned herbal tonics. It is used as general tonic for all kinds of weakness and to promote strength and vigour, being regarded as an aphrodisiac and rejuvenator (Agarwal et al., 1999; Dhuley, 2000; Ziauddin et al., 1996). Various parts of the plant have been reported to possess antiserotogenic, anticancer (Anon, 2004; Jayaprakasam, 2003; Mathur, 2006) and anabolic properties and has beneficial effects in the treatment of arthritis, stress and geriatric problems (Asthana and Raina, 1989; Gandhi et al., 1994; Devis and Kuttan, 2000; Singh et al., 2001; Prakash et al., 2002). A mosaic disease was observed on W. somnifera during the surveys in at Aligarh and Lucknow in Uttar Pradesh and Hindaun City in Rajasthan, India. The disease incidence was significant (15-25%). The naturally infected W. somnifera plants exhibited yellow mosaic accompanied with yellow net, vein clearing, vein thickening, vein spreading and mild leaf curl symptoms. Since W. somnifera is a very important medicinal plant in India and the incidence of mosaic disease on W. somnifera was significant which affected adversely to its yield and biomass, therefore, it was worthwhile to investigate the virus associated with the mosaic disease so that some control measures may be suggested to the growers of this plant. 5.1 Biological Characterization The characteristic disease symptoms (mosaic and leaf curl) indicated the possibility of geminivirus infection as the symptoms showed resemblance with that of Jatropha mosaic disease caused by Jatropha mosaic India virus (Aswatha Narayana et al., 2007) and the mosaic on tomato caused by Tomato yellow vein 101

3 streak virus (Colariccio et al., 2007) that belong to the Geminiviridae family. The positive results of virus transmission through whitefly indicated the possibility of begomovirus occurrence on W. somnifera as whiteflies (B. tabaci) are the vectors of begomoviruses (Fauquet et al., 2008) such as Tobacco leaf curl virus (Storey, 1931), Tomato leaf curl virus (Muniyappa et al., 2000), Indian cassava mosaic virus (Antony et al., 2006), Ageratum yellow vein virus (Bigarre et al., 2001), African cassava mosaic virus (Storey and Nichols, 1938), Potato yellow mosaic virus (Coutts et al., 1991). Further, the presence of twinned, icosahedral virus particles of nm in diameter during TEM studies detected begomovirus association with mosaic disease of W. somnifera as such type of particles of Tomato leaf curl virus were also detected through TEM (Anjaneya Reddy, 2006). The host range study indicated that the virus under study consisted of narrow host range and transmitted mostly on solanaceous plants. Parthenium hysterophorus was also found as experimentally inoculated host. This finding was similar to that of Tobacco leaf curl virus as P. hysterophorus was a host plant of this begomovirus. Host range studies are important for the virus purification and maintenance of virus culture. It is also essential for devising a possible management strategy to the virus causing disease in W. somnifera. Literature revealed occurrence of Tobacco leaf curl virus (Phatak and Raychaudhuri, 1967) and Eggplant mottled dwarf virus (Al-Musa and Lockhart, 2008) on W. somnifera. Al- Ani in 1994 reported Cucumber mosaic virus on W. somnifera. However, begomovirus causing mosaic has not been reported earlier from any part of the world on W. somnifera. The begomovirus associated with mosaic disease of W. somnifera was detected initially on the basis of transmission through whitefly, electron microscopic study. 5.2 Molecular characterization Polymerase chain reaction by using degenerate begomovirus primers showed positive amplicons of expected size ~800 bp and ~1.2 Kbp DNA bands on gel electrophoresis from symptomatic samples confirmed begomovirus infection. Further, the association of begomovirus with mosaic disease of Withania somnifera was detected at three locations in India by PCR using begomovirus specific primers. The resulting amplicons of ~800 bp using TLCV-CP primers 102

4 (Hallan, 1998) and ~1200 bp using Rojas primers (Rojas, 1983) of three locations were sequenced and sequence data was analyzed. The CP gene of these isolates shared 91-94% identity with Jatropha mosaic India virus (HM230683) based on BLASTn analysis. Analysis by pairwise percent identity revealed that the Withania isolates shared 86-92% identity at nucleotide and 95-98% identity at amino acid levels with JMIV. Phylogenetic tree clearly revealed the closest relationship with JMIV at both nucleotide and amino acid levels of all the three Withania isolates. Same results were repeated when the sequence of ~1.2 kb amplicon was analyzed. These isolates shared % identities among them and 91-93% identity with Jatropha mosaic India virus (JMIV) and 84-85% identities with isolates of Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) on the basis of BLAST analysis.based on the highest sequence identities and close phylogenetic relationships with Jatropha mosaic India virus (JMIV), the begomovirus isolate associated with the mosaic disease of W. somnifera were identified as isolates of JMIV under the group of begomovirus, genus Geminivirus and family Geminiviridae. To confirm, whether the virus isolate is monopartite or bipartite, the PCR was also performed using BC1F and BC1R primers located in DNA-B (Padidam et al., 1995) and β01and β02 primers (Briddon et al., 2003). However, the PCR carried out with DNA-B specific primers and DNA-β specific primers did not result positive amplification in any of the plant samples, confirming the virus isolate to be monopartite and absence of association of DNA-β molecule with the disease. The association of begomovirus with yellow mosaic disease of W. somnifera has been reported earlier on the basis of virus transmission by whitefly, electron microscopic observations and positive amplification of expected size DNA band by PCR using begomovirus specific primers (Baghel et al.,, 2010). Jatropha mosaic India virus is the first non-cassava-infecting begomovirus that is highly related to cassava mosaic viruses (Aswatha Narayana et al., 2007). However, molecular identification and characterization of the JMIV isolate associated with mosaic disease of W. somnifera is a new report from India. 103

5 5.3 Pathological effects of begomovirus isolate under study on W. somnifera Geminiviruses pose a serious threat to the cultivation of many agriculturally important crops, particularly in the tropics and sub-tropics (Varma and Malathi, 2003; Prasanna and Rai, 2007). Therefore, the studies were conducted to measure the impact of begomovirus infection on host plant s health. Study on growth and biomass yield of W. somnifera indicated significant (Baghel et al., 2010). The study suggests that there is a correlation between virus infectioninduced morphological and anatomical alterations with the shift in metabolic profiles. Recently, it was found that infection of Tomato yellow mosaic virus in tomato caused severe symptoms which included yellowing, reduction of the leaf lamina and stunting of the plant (Leal and Lastra, 2008). These findings are similar to that of our results in terms of height and weight of the virus infected W. somnifera. Jatropha mosaic India virus-[withania] reduces the leaf quality and leaf area like African cassava mosaic virus (Thresh et al., 1994). When healthy and infected plants were compared, a reduction in chlorophyll contents of W. somnifera was evident in infected leaves. The deleterious effects are probably associated with the impaired efficiency of leaves affected by mosaic and various changes have been reported in chloroplast structure, chloroplast number and chlorophyll contents (Chant and Beck, 1959; Chant et al.,, 1971; Ayanru and Sharma, 1982). The physiological and biochemical alterations in the tissues and changes in metabolic processes within the plant act as a defense mechanism against virus infection (Fraser, 1987). These observations indicated the requirement of proper management practices of the present virus isolate for qualitative and quantitative improvement in the production of W. somnifera. Thus, the interesting outcome of the present study is that the sequence data generated out of the study would be helpful in designing the begomovirus specific primers for quick and reliable detection of begomovirus so that the population of W. somnifera may be screened to find out the virus free and healthy plant materials for its related pharmaceutical industries. The sequence data generated on the coat protein gene of the begomovirus may be utilized for genetic transformation of W. somnifera to develop the begomovirus resistant transgenic plants and the production may be enhanced by several folds. 104