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Endochitinase Activity of Trichoderma reesei to Suppress Fungal Root Rot Pathogen Ganoderma philippii.
Abstract:
Ganoderma philippii (Bres. & P. Henn.) Bres, is a potential root rot disease agent causing serious damage in many types of tree plantation. Of the methods developed to control the disease, none gives successful result. Trichoderma known as having mycoparasitic action against fungal plant pathogens has been applied as biocontrol agent against Ganoderma root rot disease in some estate plantations. However, the mycoparasitic mechanism of Trichoderma against fungal plant pathogen is not fully described. This experiment aimed to determine the endochitinase activity of Trichoderma against root rot pathogen, G. philippii.
Extracellular endochitinase was produced by growing mycoparasite T. reesei strain T13 in chitin as sole carbon source. The enzyme was then purified to its homegeneity by precipitation with ammonium sulfate, followed by gel filtration chromatography and chromatofocusing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE [12.5%]) was used to check the purity of enzyme at each stage of preparation and to characterize purified protein. Fungal bioassay was done using modified cup plate method.
The results showed that the enzyme has molecular weight of 32 kDa and optimum activity at pH of 5.5 and temperature of 30-35°C. Bioassay to test antifungal activity of endochitinase against the mycelia of G. philippii indicated that the degree of inhibition was proportional to the level of enzyme concentration applied. Inhibition zone occurred macroscopically at the outer line of the colony of G. philippii at concentration of enzyme 80 μg ml-1 and above. The enzyme caused necrotic lesion on mycelial cell wall and branching responses of mycelial tip at the concentration of 60- 200 μg ml-1. At the concentration of 100-200 μg ml-1, endochitinase lysed the hypha of G. philippii, indicated by coiling around the hypha of G. philippii. T. reesei’s hypha was noted in the experiment.