ENDOCHITINASE FROM GLIOCLADIUM-VIRENS - ISOLATION, CHARACTERIZATION, AND SYNERGISTIC ANTIFUNGAL ACTIVITY IN COMBINATION WITH GLIOTOXIN

The culture filtrate from the biocontrol agent Gliocladium virens strain 41 grown in chitin-containing medium was strongly inhibitory to mycelial growth of different plant-pathogenic fungi. The antibiotic gliotoxin was isolated from the culture liquid. The culture filtrate also contained different types of chitinolytic enzyme activities, including endochitinase, chitin 1,4-beta-chitobiosidase, and glucan N-acetyl-beta-D-glucosaminidase, as well as glucan 1,3-beta-glucosidase activity. An endochitinase was purified to homogeneity. The enzyme had a molecular weight of approximately 41,000 Da and a pI of 7.8. The optimal range for enzyme activity was pH 4-6. The inhibitory effect of pure endochitinase and gliotoxin on the germination of conidia and germ tube elongation of Botrytis cinerea was tested in vitro. When applied alone at concentrations of 150 mug ml-1, the endochitinase inhibited spore germination of the test fungus and caused cell wall damage, resulting in bursting of hyphal tips. The ED50 value of gliotoxin was 1.25 mug ml-1. When gliotoxin and the endochitinase were applied together, a synergistic inhibitory effect was observed. Addition of 25 or 50 mug ml-1 of endochitinase reduced the ED50 of gliotoxin to 0.75 mug ml-1 and 0.5 mug ml-1, respectively. These enzyme concentrations applied alone showed no inhibitory effect. Furthermore, applied individually, 0.75 mug ml-1 of gliotoxin and 75 mug ml-1 of endochitinase caused no inhibition and 20% inhibition, respectively, whereas the combined application resulted in 95% inhibition. Synergistic antifungal activity of endochitinase and gliotoxin may play a role in biological control by G. virens.