PARALLEL FORMATION AND SYNERGISM OF HYDROLYTIC ENZYMES AND PEPTAIBOL ANTIBIOTICS, MOLECULAR MECHANISMS INVOLVED IN THE ANTAGONISTIC ACTION OF TRICHODERMA-HARZIANUM AGAINST PHYTOPATHOGENIC FUNGI

Chitinase, beta-1,3-glucanase, and protease activities were formed whe n Trichoderma harzianum mycelia, grown on glucose as the sole carbon s ource, were transferred to fresh medium containing cell walls of Botry tis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by We stern blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenou sly added [U-C-14]valine was incorporated in acetone-soluble compounds with an apparent M(r) of <2,000. These compounds comigrated with the peptaibols trichorzianines A(1) and B-1 in thin-layer chromatography a nd released [U-C-14]valine after incubation in 6 N HCl. Incorporation of radioactive valine into this material was stimulated by the exogeno us supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inh ibited spore germination as well as hyphal elongation of B. cinerea. C ulture supernatants from mycelia placed in fresh medium without cell w alls of B. cinerea did not show hydrolase activities, incorporation of [U-C-14]valine into peptaibol-like compounds, and inhibition of funga l growth. Purified trichorzianines A(1) and B-1 as well as purified ch itobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than t hose observed in the culture supernatants. However, when the enzymes a nd the peptaibols were tested together, an antifungal synergistic inte raction was observed and the 50% effective dose values obtained were i n the range of those determined in the culture supernatants. Therefore , the parallel formation and synergism of hydrolytic enzymes and antib iotics may have an important role in the antagonistic action of T. har zianum against fungal phytopathogens.