Cell wall synthesis is a major target of mycoparasitic antagonism by Trichoderma harzianum

Abstract

We have investigated the molecular basis for the reported synergism between peptaibols and cell wall hydrolytic enzymes in the antagonism of phytopathogenic fungi by Trichoderma harzianum. ␤-Glucan synthase activity on isolated plasma membranes of Botrytis cinerea was inhibited in vitro by the peptaibols trichorzianin TA and TB, and this inhibition was reversed by the addition of phosphatidylcholine. ␤-Glucan synthesis in vivo, assayed by the incorporation of [2-3 H]glucose into cell wall material, was inhibited by the presence of peptaibols, and this inhibition was synergistic with exogenously added T. harzianum ␤-1,3-glucanase. This synergism is therefore explained by an inhibition of the membrane-bound ␤-1,3-glucan synthase of the host by the peptaibols, which inhibit the resynthesis of cell wall ␤-glucans, sustain the disruptive action of ␤-glucanases, and all together enhance the fungicidal activity. Therefore, we have identified cell wall turnover as a major target of mycoparasitic antagonism.The soil fungus Trichoderma harzianum is active against a range of economically important aerial and soilborne plant pathogens and is successfully used as a biopesticide in greenhouse and field applications (4, 23). The antagonistic mechanism of T. harzianum is a complex process involving chemotropism (5), lectin-mediated recognition (15-17), and formation of trapping and penetration structures (7,8). This process is further supported by the secretion of extracellular enzymes such as chitinases (3,6,14), ␤-glucanases (13, 19, 21), and proteinases (11) as well as secondary metabolites (1, 2, 12). However, the fungicidal mechanism of T. harzianum is so far unknown. A key to understanding this mechanism of antagonism is the recently found synergism between hydrolytic enzymes and membraneaffecting antibiotics such as peptaibols (20,24) which lowers the active concentrations of the two types of compounds to the level observed in vivo. Peptaibols are linear amphipathic and antibiotic 7-to 20-residue peptides, mainly produced by fungi of the genus Trichoderma. They are structurally characterized by an acylated N-terminal residue, a C-terminal amino-alcohol (tryptophanol or phenylalaninol), and a high content in ␣-aminoisobutyric acid. They have been shown to interact with phospholipid bilayers and to increase their permeability for concentrations ranging between 10 Ϫ6 and 10 Ϫ7 M (10, 18). They form voltage-gated ion channels in planar bilayers at 10 Ϫ8 M (22). When combined with hydrolytic enzymes, they produce synergistic mixtures with strong inhibitory activity on the growth of many pathogenic fungi (20, 24). We therefore speculated that the synergism of peptaibols with chitinases and ␤-glucanases could be due to an interference with cell wall synthesis associated with the plasma membrane.We have first tested this hypothesis in vitro by using trichorzianins TA and TB from T. harzianum and ␤-glucan synthase from three different fungi: Botrytis cinerea, T. harzianum, and Saccharomyces cerevisiae. Trichorzianin TA is a micro...

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