Endo-1,3-beta-glucanase and cellulase from Trichoderma harzianum: Purification and partial characterization, induction of and biological activity against plant pathogenic Pythium spp.

There were indications that endo-1,3-beta-glucanase (1,3-(1,3;1,4)-beta-D-Glucan 3(4)-glucanohydrolase (EC 3.2.1.6)) and cellulase (1,4-(1,3;1,4)-beta-D-Glucan 4-glucanohydrolase (EC 3.2.1.4)) activity of Trichoderma harzianum Rifai isolate T3 were induced in sphagnum peat moss cultivations and dual culture experiments by the presence of Pythium ultimum. Further, P. ultimum stimulated the germination of Trichoderma conidia. Endo-1,3-beta-glucanase and cellulase were purified from T. harzianum isolate T3, known to control Pythium damping-off of cucumber seedlings. The enzymes were purified from the culture filtrate of the fungus by gel filtration and isoelectric focusing. The purified endo-1,3-beta-glucanase was a small protein with a molecular mass of 17 kilodaltons and a pi of 5.0. Two cellulases were purified to homogeneity and had molecular masses of 40 and 45 kilodaltons respectively, and pi's of 6.4 and 7.6 respectively. Germination of encysted zoospores and elongation of germ tubes of a plant pathogenic Pythium isolate were inhibited by low concentrations of the purified enzymes. A strong synergistic effect was observed on the inhibition of cyst germination by a combination of the endo-1,3-beta-glucanase and the fungicide Fongarid. Finally, a time-course study of colonization of the rhizosphere of cucumber seedlings showed that the active fungal mycelial biomass of a GUS-transformant of T. harzianum isolate T3 increased over four weeks. Trichoderma appeared to colonize healthy roofs only superficially, whereas the mucilage of the root hairs and of distal parts of wounded areas or broken parts of the roots, were extensively colonized.