**2.5 Influence of artificial media on the activity of extracellular protease Pr1**

The activity of Pr1 protease bound to 10 mg spores harvested from the rice of above mentioned media were assayed using a modified method of St. Leger et al. (1987). Briefly, 10 mg spores were washed once in 0.3% aq Tween 80 solution and twice in distilled water, were then incubated in 1 ml of 0.1M Tris HCl (pH 7.95), containing 1mM Succinyl-ala-alapro-phe-*p*-nitroanilide (Sigma) for 5 min at room temperature. The spores were pelleted by centrifugation for 5 min at 12000 g (Fastwin Bio-Tech Company Limited). A 200 µL of supernatant was transferred to quartz cuvette. The absorbance was measured at 405 nm by using a spectrophotometer (Shimadzu UV-1800). Buffered substrate was used as a reference. The amount of spore bound extracellular protease Pr1 is expressed as micromoles of nitroanilide (NA) released per minute.
