Colletotrichum acutatum, a fungal pathogen that causes soft rot in fruit, produced protease when grown on casein or apple cell wall, as revealed by a clear zone around the well filled with C. acutatum medium in a radial diffusion assay. A protease-inhibiting protein (PI) was also extracted from healthy stored apple, cultivar Cripps Pink, and its activity was tested in vitro and in vivo against a protease produced by C. acutatum. In in vitro trials the inhibition rate determined by radial diffusion assay was over 41% after 24 h, while in inoculated fruit the inhibition ranged from 23.5% to 45% after 5 days at 20 degrees C. The protease inhibitor extracted from healthy apple skin was a heat-denaturable protein since the halo produced by protein extracted from C. acutatum and added to boiled protein extracted from healthy apple skin tissue was 338.7 mm(2), significantly higher than the halo produced by protein extracted from C. acutatum diluted with fresh protein extracted from healthy tissue (220.7 mm(2)). Protein secreted by C. acutatum grown in induced buffer media was tested by polyacrylamide gel electrophoresis. SDS-PAGE of crude enzyme extract revealed the presence of various protein bands that could be ascribed by their molecular mass as putative aspartic proteinase, extracellular alkaline proteinase and serine protease. Genomic analyses are, however, in progress to identify the proteins involved in Colletotrichum patogenicity. More investigations are required to identify the nature of the substance responsible for C. acutatum inhibition in apple and to evaluate the possibility of manipulating the protease inhibitor levels in fruit to reduce soft rot caused by C. acutatum. (C) 2010 Elsevier Ltd. All rights reserved.
