Streptomyces albidoflavus NRRL B-16746 secreted three types of chitinolytic enzymes: N-acetyl-glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4-6; however 55-74% of the chitobiosidase and endochitinase activity was detectable at pH 8-10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8-5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4-10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.

A purified preparation of trypsin and chymotrypsin inhibitors from cabbage foliage showed antifungal activity in vitro. The inhibitors suppressed spore germination and germ tube elongation of two species of phytopathogenic fungi but had no effect on a fungus that specifically attacks cabbage. Microscopic examination indicated that the inhibitors caused leakage of the intracellular contents from the susceptible species of fungi.

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.

Trichoderma harzianum strain P1 produces a variety of chitinolytic enzymes including N-acetyl-β-D-glucosaminidases, chitin 1,4-β-chitobiosidases, and an endochitinase. Chitabiosidases and an endochitinase were purified from dialyzed, concentrated culture filtrates using gel filtration, chromatofocusing, and isoelectric focusing. Three protein bands were evident in the purified chitobiosidase preparation, representing different levels of N-glycosylation of the same protein. The pI of all purified proteins was ∼3.9 []

Two chitinolytic enzymes from Trichoderma harzianum strain P1 were tested for their antifungal activity in bioassays against nine different fungal species. Spore germination (or cell replication) and germ tube elongation were inhibited for all chitin-containing fungi except T. harzianum strain P1. The degree of inhibition was proportional to the level of chitin in the cell wall of the target fungi. For most of the fungi tested, the ED50 values for the endochitinase and the chitobiosidase were 35-135 mug ml-1 and 62-180 mug ml-1, respectively. Complete inhibition occurred at 200-300 mug ml-1. Combining the two enzymes resulted in a synergistic increase of antifungal activity. The ED50 values for a 1:1 mixture of endochitinase and chitobiosidase were as low as 10 mug ml-1 for Botrytis cinerea, 34 mug ml-1 for Ustilago avenae, 13 mug ml-1 for Uncinula necator, and 30 mug ml-1 for Fusarium solani. T harzianum strain PI was resistant to its own chitinolytic enzymes up to 800 mug ml-1, with an ED50 value>1,000 mug ml-1. The chitinolytic enzymes from T. harzianum appeared to be biologically more active than enzymes from other sources and more effective against a wider range of fungi. The involvement of these chitinolytic enzymes in biocontrol is also discussed.