A drastic increase of antifungal activity was demonstrated during plant seed germination and in seed protein extract in vitro. Multiple antifungal proteins with a wide spectrum of activity were generated and identified. Chromatographic and electrophoretic analysis demonstrated that during seed germination, more fractions with potent antifungal activity were generated, and the antifungal activity shifted from small molecules to high molecular proteins. This germination-related increase of antifungal activity were observed in all three plants tested, i.e., cheeseweed, cigar tree and wheat. This rapid increase of antifungal activity was also observed with incubation of seed proteins in vitro, suggesting that at least part of the antifungal protein generation is independent of gene expression. Seven antifungal proteins with activities against five different plant pathogens were isolated from the active fractions. However, random digestion of purified seed protein with multiple proteinases failed to generate any antifungal proteins. It is suggested that during plant seed germination, a regulated biochemical process takes place that results in the generation of multiple peptides or proteins with antifungal activities. This onset of antifungal proteins is transitional in nature, but could play an important role in the protection of plants in early stage of development when the more sophisticated defense system has yet to develop. (C) 2002 Elsevier Science (USA).