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For estimating enzyme activities 1g of leaf tissue sample was extracted by homogenizing in 10 ml of cold 0.1 M phosphate buffer, pH 7.0 containing 1 mM cysteine hydrochloride and 0.1% ascorbic acid in a chilled pestle mortar using acid washed sand as an abrasive. The homogenate was filtered through four layers of cheese cloth and subsequently the filtrate was centrifuged at 15,000 g for 20 min in a refrigerated centrifuge at 4oC The supernatant obtained was used for enzyme activity assays.

Polyphenoloxidase (EC 1.10.3.2) was assayed following the method of Taneja and Sachar (1974). The reaction mixture contained 2.0 ml of 1% catechol solution as substrate, 0.2 mI of enzyme extract and rest of 0.05 M phosphate buffer pH 6.6 in a final volume of 4 ml. Boiled enzyme extracts served as the control. The change recorded at 430 nm was expressed for enzyme activity as change in absorbance per mg of protein per hour. For estimation of catalase (EC 1.11.1.6) activity the assay method of Beers and Sizer (1962) was followed. During activity assay the sample cuvette contained in a final volume of 3.0 ml; 0.1 mI of 0.2 tissue extract, 0.5 ml of 0.2 M sodium phosphate buffer pH 7.6, 0.3 ml of hydrogen peroxide and rest of distilled water. The enzyme activity was expressed as the change in absorbance per min per mg protein when measured at 240 nm.

For estimation of leakage of amino acids and UV-absorbing materials equal number of leaves (32) weighing almost equal (4.0 g) were taken. Each leaf was cut into four equal pieces and were put into 100 ml of deionized water in a 250 ml capacity conical flask and were shaken at 70 strokes per min for 8 hours in a water bath at 37oC. The leaves were then filtered out and in the filtrate the concentration of amino acids was determined as glycine equivalents by taking 0.5 mI of the filtrate as per the method of Barnett and Naylor (1966). For UV-absorbing materials the O.D of the filtrate was read at 270 nm.


Results and discussion

The enzymatic activities (Table1) of polyphenoloxidase and catalase was found to be increased as a result of progressive rust infection upto 36h in both susceptible (race 77) and resistant (race 63) - interactions. However the increase was more in susceptible-interaction due to race 77. Toward later stages (ie at 48h and 72h), polyphenoloxidase activity declined which was found to be more in the susceptible-interaction. The activity of catalse also declined toward 48h stage but again reflected an increasing trend toward final stage of 72h. The overall activity profile remained higher in case of race 77-interaction as shown in Table1.

Higher levels of polyphenoloxidase activity in resistant~interaction particularly toward later stages of infection account for such pathophysiological conditions due for easier disease progression and its establishment. As the activity of this enzyme in diseased or wounded tissues has been shown to be accompanied by enhanced levels of phenolics which have been thought to be metabolically implicated in disease resitance phenomenon (Farkas and Kiraly 1958, Farkas and Kiraly 1962, Kosuge 1962, Rohringer and Samborski 1967, Bell 1981, Goodman et al 1986). The higher activity profile of catalase in the susceptible-interaction signifies the differential race pathogenesis as the increased levels of catalase endangered by virulent isolets have been shown to reduce the efficacy of natural defence of the bean plants against Pseudomonas phaseolicola through supression of peroxidase activity by destroying its substrate hydrogen peroxide. Thus creating a redox potential environment believed to favour the maintenance of phenolics in their reduced state which are considered less active as antimicrobial substances than their quinone forms (Rudolph and Stahmann 1964). In general occurence, of the lowered activity of polyphenoloxidase and the elevated activity of catalase, in the susceptible-interaction due to race 77 and that too particularly toward later stages of progressive rust infection characterizes the general nature of infection in this system.

Comparatively less leakage of amino acids in case of resistant-interaction (Table 2) may be due to immediate defensive host response toward the pathogen by trying to make less available the metabolites for the growth of the pathogen or may be due to such related changes as result of attempted infection. Alternatively, the comparatively increased leakage in case of susceptibleinteraction may either be due to adaptive cointeraction or due to disintegration of the host cellular membranes (Saini et al 1989, Saini et al 1990). The higher losses of the metabolites particularly at later stage of Uromyces infection of the Phaseolus leaves might be due to presence of fungal mycelium and spores (Hoppe and Heitefuss 1974). The decreased leakage of UV-absorbing materials through successive stages of infection in susceptible-interaction (Table 2) probably be taken for their degradation first and then consumption of such substances for anabolic purpose by the growing pathogen to make them less available to leak. The physiological changes during progressive rust infection signifies at least partial correlations to susceptible and resistant pathogenesis of wheat leaves during its early course upto 72h stage.

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