Browsing by Author "Thambugala, T.A.N.S."
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Item Novel poly amine biosynthetic inhibitors to control the phytopathogenic fungal diseases(University of Kelaniya, 2008) Weerasooriya, M.K.B.; Thambugala, T.A.N.S.Fungal polyamines play a major role in controlling the growth and development of the fungal cells. Higher polyamine levels enhance the fungal growth whereas lower levels retard the growth. Therefore depleting polyamine level by selective inhibition of poly amine biosynthesis through specific inhibitors, could be used to control variety of fungal diseases in plants. Following this line of thinking we aimed to identify potential and specific inhibitors of polamine biosynthetic enzymes. The fungus Colletotrichum gleosporoides which causes anthracnose in a wide range of plant in many parts of the world was selected for the study. Arginine decarboxyalse, a rate limiting enzyme of the polyamine biosynthesis was isolated from fungus. Fungus was grown in Cook's 2 media. Crude enzyme extract was prepared from 12-13 days old cultures, mycelia were harvested, immersed in citrate buffer (50mM, pH 5.6) and ground in a motor with a pestle at 4°C. Thus prepared crude enzyme extract was partially purified 25 fold with 16.7% recovery by ammonium sulphate fractionation (0-25% ) followed by DEAE cellulose and gel filtration chromatography. Enzyme activity was assayed by detecting the released carbon-dioxide with warbug manometer. Several plant extracts were tested as possible inhibitors for the enzyme. Of them Leaf extract of Ocimum sanctum (maduru thala) and Jatropha curcus (Rata Endaru) and tuber extract of Acarus calamus (Wadha kaha) and Zingiber zerumbet (Wal inguru) were found as inhibitors for the enzyme where as leaf extract of Ocimum basillicum (Suwanda thala) and tuber extract of Costus speciosus (Thebu) were found as non inhibitors for the enzyme. 1 00% inhibition of the enzyme was seen at 0.1 g/ 1 ml concentration level of Ocimum sanctum and Jatropha curcus and 0.08g/lml level of Acarus calamus, and Zingiber zerumbet.Item Ornithine decarboxylase from Colletotrichum gloeosporioides: its purification, properties and inhibitors(Sri Lanka Association for the Advancement of Science, 2011) Weerasooriya, M.K.B.; Thambugala, T.A.N.S.Fungal polyamines play an indispensable role in controlling the growth and development of fungal cells. High levels of polyamines enhance cell growth where as low levels retard growth. Therefore, depleting polyamine levels by selective inhibition of polyamine biosynthesis by specific inhibitors will provide a key to controlling a variety of fungal diseases in plants. With this aim, Ornithine decarboxylase, a rate limiting enzyme of fungal polyamine biosynthesis, was isolated from the fungus Colletotrichum gloeosporioides, and purified by ammonium sulphate fractionation followed by DEAE cellulose and sepharose 4B gel filtration chromatography. The enzyme was purified 11.56 fold with 58 % recovery. The purity of the enzyme fraction was screened by SDS-PAGE. Two bands were seen at ~65kDa and ~ 25 kDa. The native molecular mass of the enzyme as determined by sepharose 4B gel filtration chromatography was ~ 130KDa. Kinetic studies of the enzyme reaction showed that enzyme exhibited higher activity in the range of 35-45°C with a maximum at 40°C. Effect of pH on enzyme activity showed that the enzyme possesses high activity in the range of 4.8- 5.4 with an optimum at pH 5.2. Inhibition studies with the enzyme showed that eugenol, linalool, α-pinene, geraniol , citral were not inhibitors. Cyclohexylamine, methyl isoleucine, methyl isothiourea and methyl hydrazine carboxylate act as inhibitors for the enzyme.Item Screening of biofungicides to control the fungal diseases caused by Colletotrichum gloeosporioides(2010) Thambugala, T.A.N.S.