Molecular Genetic Basis Of Biocontrol

Arising from nectrotrophic ancestors, most of the currently known Trichoderma strains have developed highly effective antagonistic mechanisms to survive and colonize the basidiomycete-containing competitive environment of the rhizosphere, soil, and decaying wood. Active parasitism on host fungi, by penetration of host hyphae is probably the mechanism most studied (cf. Chet et al. 1998); it requires morphological changes of Trichoderma hyphae such as appressorium formation and coiling, and is further supported by the production of extracellular enzymes, and production of antifungal antibiotics. However this mechanism has mostly been observed in the laboratory, and application of Trichoderma in the field may involve additional mechanisms as well such as aggressive degradation of organic matter, thereby competing for nutrients which in saprobic phases may be a limiting factor. Also promotion of the growth and biological activities of saprobic bacteria and mycorrhizal fungi, and of plant-growth and induced resistance have been reported (for review see Herrera-Estrella and Chet, Chapter 57).

Among these, mycoparasitism is the only process which has been studied on a molecular biological basis. Herrera-Estrella and Chet (Chapter 57) give a detailed account on this, and I shall therefore treat this point here only very briefly: most attention has been paid to the enzymatic disruption of the cell wall of the fungus, thereby focusing on enzymes capable of hydrolysis its structural polymers (chitin, b-glucan, protein and others). Genes encoding endochitinases, N-acetyl-b-glucosaminidases, proteases, endo- and exo-glucan b-1,3-glucosidases, endoglucan-b-1,6-glucosidases, lipases, xylanases, amylases, phospho-lipases, RNAses, and DNAses have been cloned from various biocontrol species of Trichoderma, and are listed in detail in the above-mentioned chapter (also see Benitez et al. 1998; Kubicek et al. 2001; Lorito 1998). Most of these enzymes showed very strong antifungal activity against a variety of plant pathogenic fungi in vitro. Several of these cell wall degrading enzymes, but most notably chitinases, have thus been demonstrated to have a great potential as active components in new fungicidal formulations or genetically modified plants.

Interestingly, the endochitinases found in Trichoderma belong only to one (class V) of the several classes of the chitinases known from plants (Beintema 1994). The latter show a modular structure, and frequently contain protein domains capable of binding to chitin, which bear some resemblance to the cellulose-binding domains also found in Trichoderma cellulases. In contrast, none of the chitinases cloned from Trichoderma spp. so far has been shown to contain such a chitin-binding domain. To investigate the role of the latter, Limon et al. (2001) have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of T. reesei. The chimeric chitinases had similar activities as the native chitinase towards soluble substrates, but higher hydrolytic activity on high molecular mass insoluble substrates (chitin or fungal cell walls). Unfortunately, no results from in vivo biocontrol tests were reported, and it remains thus unclear whether the presence of such a domain would improve the antagonistic abilities of Trichoderma biocontrol strains.

The action of chitinases and glucanases is also strongly synergistic both with other chitinase components as well as with other components putatively involved in biocontrol, i.e., antibiotics (Jach et al. 1995; Lorito et al. 1994; 1996b; Schirmbock et al. 1994). In the case of the peptaibols, the mechanism of this enzyme-antibiotic synergism has been shown to be due to a synergistic effect of enzyme and the antibiotic on the maintenance of cell wall integrity (Lorito et al. 1996b). Peptaibols are linear oligopeptides of 12-22 amino acids, which are rich in a-aminoisobutyric acid, N-acetylated at the N-terminus and containing an amino alcohol (Pheol or Trpol) at the C-terminus (Rebuffat et al. 1989), and known to form voltage-gated ion channels in black lipid membranes and modify the membrane permeability of liposomes in the absence of applied voltage (El Hadjji et al. 1989). Hence, while the chitinases reduce the barrier effect of the cell-wall, peptaibol antibiotics inhibit the membrane bound chitin- and b-glucan synthases and thereby impair the ability of the hyphae to repair the lytic effect of the enzymes on the cell walls polymers.

The gene (tex1) encoding the enzyme synthesizing these peptaibols (peptaibol synthase) has recently been cloned from T. virens (Wiest et al. 2002). It comprises a 62.8 kb continuous open reading frame encoding a protein structure consisting of 18 peptide synthetase modules with additional modifying domains at the N- and C-terminii. Mutation of the gene eliminated production of all peptaibol isoforms, indicating that their formation is due to a relaxed substrate specificity of the individual synthase domains. Interestingly, the nucleotide sequence of tex1 is 100% identical to a 5,056-bp partial cDNA fragment of another gene (psyl) isolated also from T. virens (Wilhite et al. 2001). These authors observed that psy1 disruptants grew poorly under low-iron conditions, and failed to produce the major T. virens siderophore, dimerum acid (a dipeptide of acylated N(a)-hydroxyornithine, thus suggesting that Psy1 plays a role in siderophore production. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the psy1disruption. The discrepancy between the results reported by Wiest et al. (2002); Wilhite et al. (2001) need to be explained before the importance of the tex1/psy1 gene in biocontrol can be estimated.

Peptaibols, however, are certainly not the only secondary metabolites with synergistic action in host cell-wall degardation. Other components (e.g., gpentyl pyrone) was also found to be important for antagonism in vivo (Claydon et al. 1987; Howell 1998; Serrano-Carreon et al. 1993), and their mechanism of action thus awaits to be elucidated. 6-pentylapyrone is probably the most frequently studied of these metabolites, as it also exhibits a pronounced "coconut-aroma" which can be used as a (for humans) nontoxic flavoring agent. Its biosynthesis has been claimed to be derived from linolenic acid (Serrano-Carreon et al. 1993), but this conclusion was criticized by Sivasithamparam and Ghisalberti (1998), who consider it to be a product of polyketide biosynthesis. No other of the genes or proteins involved in Trichoderma secondary metabolism has as yet been characterized.

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