Detection of sandal spike phytoplasma using immunological and molecular techniques

Spike disease in sandal is generally diagnosed by the manifestation of external symptoms. Attempts have been made to detect the diseased plants by determining the length/breadth ratio of leaves (lyengar, 1961) and histochemical tests using Mann's stain (Parthasarathi et al., 1966), Dienes' stain (Ananthapadmanabha et a/., 1973) aniline blue and Hoechst 33258 (Ghosh et a/., 1985, Rangaswamy, 1995). But most of these techniques are insensitive, indirect detection methods leading to misinterpretation of results. Moreover, to identify disease resistant sandal trees, highly sensitive techniques are needed to detect the presence of the pathogen. In sandal forests, several host plants of sandal like Zizyphus oenop/ea (Fig. 1.3) also exhibit the yellows type disease symptoms. Immunological and molecular assays have to be developed to confirm the presence of sandal spike phytoplasma in such hosts. The major objectives of the present work includes:In situ detection of sandal spike phytoplasma by epifluorescence microscopy and scanning electron microscopy.,Purification of sandal spike phytoplasma and production of polyclonal antibodies.,Amino acid and total protein estimation of sandal spike phytoplasma.,Immunological detection of sandal spike phytoplasma., Molecular detection of sandal spike phytoplasma.,Screening for phytoplasma in host plants of spike disease affected sandal using immunological and molecular techniques.

Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11

Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30–80 C) and pH (7.0–10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries