Purification, characterization, and synergistic activity of a glucan 1,3-beta-glucosidase and an N-acetyl-beta-glucosaminidase from Trichoderma harzianum

A glucan 1,3-beta-glucosidase (EC 3.2.1.58) and an N-acetyl-beta-glucosaminidase (EC 3.2.1.30) were purified to homogeneity from the culture filtrate of Trichoderma harzianum strain P1. The molecular masses and the pIs were 78 kDa and 6.2, respectively, for the glucan 1,3-,beta-glucosidase and 72 kDa and 4.6, respectively, for the N-acetyl-beta-glucosaminidase. The glucan 1,3-beta-glucosidase and the N-acetyl-beta-glucosaminidase were tested against Botrytis cinerea, and their antifungal activity was compared with that obtained for an endochitinase and a chitin 1,4-beta-chitobiosidase also purified from T. harzianum strain P1. The four cell wall-degrading enzymes were also tested as mixtures containing two, three, or all four proteins in all possible combinations. A synergistic, inhibitory effect was detected on both spore germination and germ tube elongation of B. cinerea when two, three, or four enzymes were applied together. The highest level of antifungal activity was obtained when a solution containing four different cell wall-degrading enzymes was used. ED50 (50% effective dose) values were as low as 1.6 microgram ml-1 for inhibition of conidial germination and 1.7 microgram ml-1 for inhibition of germ tube elongation of the surviving spores