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Molecular Plant Pathology

Purification, Characterization, and Synergistic Activity of a Glucan 1,3-β-Glucosidase and an N-Acetyl-β-Glucosaminidase from Trichoderma harzianum. M. Lorito, Department of Horticultural Sciences, Cornell University, New York State Agricultural Experiment Station, Geneva 14456, Permanent address: Istituto di Patologia Vegetale, Universitą degli Studi di Napoli and Centro CNR di Studio delle Tecniche di Lotta Biologica, 80055 Portici, Napoli, Italy; C. K. Hayes(2), A. Di Pietro(3), S. L. Woo(4), and G. E. Harman(5). (2)(3)(4)(5)Department of Horticultural Sciences, Cornell University, New York State Agricultural Experiment Station, Geneva 14456; (3)Permanent address: Departamento de Genetica, Universidad de Córdoba, 14071 Córdoba, Spain. Phytopathology 84:398-405. Accepted for publication 5 January 1994. Copyright 1994 The American Phytopathological Society. DOI: 10.1094/Phyto-84-398.

A glucan 1,3-β-glucosidase (EC 3.2.1.58) and an N-acetyl-β-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-?-glucosidase and 72 kDa and 4.6, respectively, for the N-acetyl-?-glucosaminidase. The glucan 1,3-?-glucosidase and the N-acetyl-β-glucosaminidase were tested against Botrytis cinerea, and their antifungal activity was compared with that obtained for an endochitinase and a chitin 1,4-β-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 μg ml^–1 for inhibition of conidial germination and 1.7 μg ml^–1 for inhibition of germ tube elongation of the surviving spores.