Mechanisms Involved In The Cold Tolerant Trichoderma Atroviride Biocontrol

Abstract

Trichoderma atroviride is a cold tolerant fungus that parasitizes a wide range of plant pathogenic fungi. The mechanisms involved in biocontrol by T. atroviride are only partially understood. This research evaluated the effect of four different groups of plant pathogenic fungi (Botrytis cinerea, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum) on enzyme expression at 22�C and 7�C. The enzymes expressed (proteinase and endo-beta-1,3-glucanase) were purified and characterized, and three 73 kDa N-acetyl-beta- D-glucosaminidase genes from three different T. atroviride biotypes were sequenced. The R-1,6-glucanase profiles and the regulation of N-acetyl-beta-D-glucosaminidases by plant pathogenic fungi were also studied. I document the production of N-acetyl-beta-D-glucosaminidase, exochitinase, endochitinase, beta-1,3-glucanase, beta-1,6-glucanase and proteinase by T. atroviride at room temperature. The timing of enzyme expression was pathogen dependent. A high concentration of glucose repressed the expression of glucanases, but had no effect on the expression of N-acetyl-beta-D-glucosaminidase. At 7�C, T. atroviride produced the same enzymes as at room temperature except beta-1,6-glucanase. The total activities of the chitinases increased over a 30 day incubation period while the expression of glucanases and proteinase depended on the inducer. A new 18.8 kDa serine proteinase and a new 77 kDa endo-beta-1,3-glucanase were purified to electrophoretical homogeneity. These two purified enzymes showed strong antifungal activity by inhibiting conidial germination of Botrytis cinerea. Three 73 kDa N-acetyl-beta-D-glucosaminidase genes were sequenced from T. atroviride biotypes 861, 453 and 603. Gene sequences of the enzyme from the T. atroviride biotypes are different from the published gene sequence of T. harzianum . This indicates that the N-acetyl-beta-D-glucosaminidase sequence can be used to differentiate the species and isolates of Trichoderma. The expression of beta-1,6-glucanase is complex and at least three different sizes of beta-1,6-glucanase were detected from T. atroviride. The expression of beta-1,6-glucanase varied with carbon source and pH. Mycelia of plant pathogen regulated the expression of N-acetyl-beta-D-glucosaminidase. Two different sizes of N-acetyl-beta-D-glucosaminidase were detected when T. atroviride was grown with S. sclerotiorum and its filtrates. Only one N-acetyl-beta- D-glucosaminidase was detected with other pathogens, autoclaved mycelia or glucose. The expression of a 73 kDa N-acetyl-beta-D-glucosaminidase was contact-dependent and regulated by an extracellular factor.