New components of the chitinolytic system of Trichoderma harzianum

Trichoderma harzianum is a mycoparasite known as a biocontrol agent of several economically important plant pathogenic fungi. Trichoderma spp. utilize chitinolytic enzymes, along with β-1,3-glucanase, to degrade the pathogen's cell walls and thus reduce disease level. We employed a set of three fluorescent substrates to identify chitinolytic activities of proteins renatured following their separation by electrophoresis. The chitinolytic system of T. harzianum was found to be more complex than previously reported, consisting of six distinct enzymes, two of which are described here for the first time. The system is composed of two β-1,4 N-acetylglucosaminidases (CHIT102 and CHIT73), and four endochitinases (CHIT52, CHIT42, CHIT33 and CHIT31). The newly described enzymes are CHIT73 and CHIT52. All the chitinolytic enzymes were induced and excreted during growth of Trichoderma on chitin as the sole carbon source. Only CHIT102 was expressed intracellulary, at a low constitutive level, when Trichoderma was grown on glucose. Polyclonal antibodies raised against a purified 41-kDa endochitinase produced by T. harzianum strain P1, reacted only with our CHIT42, suggesting that serologically all the other chitinolytic enzymes are not closely related to the 41-kDa endochitinase. The complexity and diversity of the chitinolytic system of T. harzianum involves the complementary modes of action of six enzymes, all of which are apparently required for maximum efficiency against a broad spectrum of chitin-containing plant pathogenic fungi.