A of the host plant, Cyamopsis tetragonoloba Taub.(Linn.).

A MARINE SEAWEED CAULERPA TAXIFOLIA Vahl. (CHLOROPHYCEAE) AGAINST TOBACCO NECROTIC VIRUS (TNV). Poonam Sethi, Asst. Prof. Guru Nanak College(Auto) Chennai, India. HYPERLINK [email protected]@rediffmail.com Caulerpa taxifolia a seaweed from the sea coast of Rameswaram, India was analysed for phytoviral activity. This seaweed was tested for activity against Tobacco necrotic virus (TNV) a virus that causes leaf necrosis and affects the yield of the host plant, Cyamopsis tetragonoloba Taub.(Linn.). Local lesion method was adopted and half leaf comparisons were made in cotyledonary leaves of Cyamopsis tetragonoloba Taub.(Linn.).Extracts of the experimental algae prepared with water , methanol and toluene were tested for antiviral activity against the plant virus TNV. Various concentrations were tested and the toluene exhibited good activity at a concentration of 1000 g/mL and reduced the number of lesion formed by TNV by nearly 64. These results clearly depict the potential of the algae abundant in the shores of Rameswaram, India during the month of April. Key words Caulerpa,Chlorophyceae, Cyamopsis , phytoviral, Tobacco necrotic virus. Plate I Cotyledonary leaves of Cyamopsis tetragonoloba showing TNV induced lesion Table – 1 Effect of the Crude Extract Residue of the Experimental Algae on the Leaves of Cyamopsis tetragonoloba infected by TNV ExtractVarying Concentrations of the Crude Extract ((g mL 1 )Mean of Number of Lesions SEP Value AQUEOUSControl 1 10 100 100022.07 0.92 20.60 0.77 15.90 0.91 10.60 0.24 10.90 0.450.000 0.070 0.000 0.000 0.000METHANOLControl 1 10 100 100023.32 0.05 18.25 0.45 15.20 0.10 12.70 0.29 11.40 0.560.000 0.003 0.000 0.000 0.000TOLUENE Control 1 10 100 100024.32 0.05 14.25 0.45 12.20 0.10 10.70 0.29 0 9.40 0.560.003 0.000 0.000 0.000 0.001 INTRODUCTION The recent increase in compounds isolated from land plants, has open doors to the poorly exploited marine ecosystem which appears to be a good candidate of natural resource Baker, J. 1984.The aquatic ecosystem covers about 70 of the earths surface and India has a vast coastline of 6100 km supporting a rich flora of marine plants such as seaweeds, mangroves and sea grasses Chapman, V. J. and Chapman, D. J,1980. As a part of our ongoing research a natural source providing mankind with various secondary metabolites having activity against viruses, which provides a new way for control of plant virus diseases. Marine algae exhibit interesting nutritional properties in addition to their ecological properties. The results of the study suggest that the algae which are abundantly available in this ecosystem also have considerable potential of carbohydrates, amino acids, proteins, phenols and lipids for their use as food and pharmaceutical industry as a source in preparation of nutrient supplements, medicine and fine chemical synthesis. As suggested by Jamieson, G. R. and Reid, E. H. 1972 the efficacy of lipid extracts was tested hence toluene extracts were used. MATERIALS AND METHOD Collection of the experimental plant and viral isolate The experimental algae was collected in the month of April from the shores of Rameswaram, India. Fresh material was shade dried and crushed in mixer to obtain a coarse powder. Local strain of the TNV (Tobacco Necrosis Virus) were obtained from Madurai Kamaraj University, Tamil Nadu. The procedures outlined by Caccamese, S et al,1981 were followed in the assay. Seeds of Cyamopsis tetragonoloba Taub.(Linn.) var. Navbahar were purchased from a government certified seed centre and surface sterilized with 0.1 acidified mercuric chloride for 3 to 5 minutes. The seeds were then sown in sterilized soil and grown in earthen pots. Preparation of various extracts Ten grams of the dried alga was extracted with 100 mL of the solvents double distilled water, methanol and toluene and later kept in a shaker for 48 hours. The three extracts were then filtered through Whatman No.1 filter paper and dried in an oven at 40oC. The residues obtained were then weighed and stored at 4oC and used for the antiviral assay. Inoculum and inoculation The inoculum was prepared by grinding 1.0 g of virus-infected cotyledonary leaves , the sap strained through cheese cloth, celite was added as an abrasive .Inoculation was carried out from the base of the leaf towards the tip with the forefinger previously dipped in the inoculum. Antiviral assay The extract residues of the experimental algae were weighed and dissolved in 0.25 DMSO (Dimethyl sulphoxide) to obtain 1.0 mg/1.0 mL concentrations. From this solution, 1 10 water dilutions were made and the inhibitory effect of these solutions were tested against tobacco necrosis virus (TNV) on the cluster bean plant, Cyamopsis tetragonoloba. Inoculations were performed on 9-day old cotyledonary leaves all the tests were made in replicates of ten, and half-leaf comparisons were made. The experimental half-leaves were inoculated with known concentrations of the extract and virus suspension. The plants were monitored for 72 hours and the number of local lesions induced by TNV was estimated using the following formula inhibition A B / A x 100 where, control-A , experimental-B Inhibition is based on the reduction of the number of local lesions induced by TNV, in the absence (A) or the presence (B) of the algal extract. The readings were taken on the basis of dark green spots of 1 mm diameter which developed as a result of infection through inoculation were observed at every 24 hour interval. The intensity of the viral infection was scored after 72 hours based on the number of lesions formed. Statiscal analysis Results were analysed for statistical significance using SPSS (Statistical Package for Scientific Studies) programme. RESULTS Extracts of the experimental algae prepared with water, methanol and toluene were tested and toluene were best in comparison to water and methanol extracts against the plant virus. The effect of the extract residues on TNV are given in Table1. At a concentration of 1000 g/mL, the toluene extract residue of Sargassum was able to reduce the lesion formation by nearly 64. DISCUSSION Earlier it was reported that the methanolic extracts of brown algae when assayed against Potato virus X inhibited the infectivity by more than 80 Pardee, K. I etal,2004, contradictory to this Kappaphycus was active against TNV with toluene (21 v/v) than methanolic extract Organic extracts obtained from the brown-alga Lessonia trabeculata inhibited bacterial growth and reduced both the number and size of the necrotic lesion in tomato leaves following infection with Botrytis cinerea whereas aqueous and ethanolic extracts from the red-alga Gracillaria chilensis prevent the growth of Phytophthora cinnamomi, showing a response which depends on doses and collecting-time. Similarly, aqueous and ethanolic extracts from the brown-alga Durvillaea antarctica were able to diminish the damage caused by tobacco mosaic virus (TMV) in tobacco leavesEdra Jimnez et al ,2011 but not much work has been done on Sargassum, has been used in herbal medicine for its anti-inflammatory, diuretic, choleretic and hemostatic properties and has been approved for food use in South eastern Asia in Japan, Korea, and TaiwanRanganayaki P etal ,2014. At present, there is a trend to use functional foods that provide health benefits by reducing the risk of chronic diseases, enhancing the ability to manage them and promoting better performance, consequently improving the quality of life. The report of this seaweed being highly active against necrotic virus was the first one. Thus it is a promising phytoviral agent. The results of the present study confirmed that definitely a significant compound is present, so further identification and purification are essential for future research. Recently marine sources are receiving much attention mainly because of functional ingredients such as polyunsaturated acids, carotene and their pigment carotenoids, sulphated polysaccharide and sterolTasende M.G., 2000. Among different compounds with functional properties, anti oxidants and antibacterials are mostly widely studiedPuthamohan Vinayaga Moorthi and Chelliah Balasubramanian2015. REFERENCES 1Baker, J. 1984. Seaweed in pharmaceutical studies and applications. Hydrobiologia 116/117 29-40. 2 Caccamese, S., Azzolina, R., Furnari, G., Cormaci, M. and Grasso, S. 1981. Antimicrobial and antiviral activities of some marine red algae from Eastern Sicily. Bot. Mar. 24 365 367. 3Chapman, V. J. and Chapman, D. J. (eds). 1980. Seaweeds and their uses. 2nd ed. Chapman Hall , New York pp.1-334. 4 Edra Jimnez , Fernando Dorta , Cristian Medina, Alberto Ramrez , Ingrid Ramrez and Hugo Pea-Corts. 2011 .Anti-Phytopathogenic Activities of Macro-Algae Extracts. Mar. Drugs. 9, 739-756. 5Jamieson, G. R. and Reid, E. H. 1972.The component fatty acids of some marine algal lipids. Phytochemistry 11 1423 1432. 6Pardee, K. I., Ellis, P., Bouthillier, M .and Towers, G. H.N. C. J . 2004.French Plant virus inhibitors from marine algae. Canadian Journal of Botany. 82(3) 304-309. 7 Pushparaj,A., Raubbin, R.S. and Balasankar, T. 2014 . Antibacterial activity of Kappaphycus alvarezii and Ulva lactuca extracts against human pathogenic bacteria Int.J.Curr.Microbiol.App.Sci ,3(1) 432-436. 8Ranganayaki,P., Susmitha, S. and Vijayaraghavan, R. 2014.Study on metabolic compounds of Kappaphycus alvarezii and its in-vitro analysis of anti-inflammatory activity. Int.J.Curr.Res.Aca.Rev,2 (10)157-166. 9 Tasende ,M.G. 2000. Fatty acid and sterol composition of gametophytes and saprophytes of Chondrus Crispus. Scientia Marina, 64(4), 421-426. 10 Puthamohan Vinayaga Moorthi and Chelliah Balasubramanian 2015. 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