Identification of gene functions involved in mycoparasitism of Trichoderma

Fungi belonging to the Trichoderma genus have received considerable attention as potential biocontrol agents due to their advantageous antagonistic properties. The mycoparasitic ability of Trichoderma spp. is well documented both against host hyphae (hyphal interference) and resting structures (sclerotia colonisation). Several strains have been proved as effective mycoparasites of many agronomically important pathogens and a number of enzymatic activities have been associated to mycoparasitic mechanisms in Trichoderma spp. The parasitic interaction established by Trichoderma against sclerotial fungi has been investigated mainly by histological experiments. Genetic engineering of biocontrol agents with reporter genes, GFP (Green Fluorescent Protein) and DsRed (Red Fluorescent Protein), has provided useful tools for monitoring antagonists in natural environments (1). A GFP T. virens transformant (I10) has been exploited for histological studies of plant pathogen/antagonist interactions, following sclerotia colonisation of Sclerotium rolfsii and Sclerotinia sclerotiorum (2). In our work mycoparasitic relations between Trichoderma and sclerotial fungi are analysed by genetic, biochemical and molecular approaches. GFP and DsRed transformants of a T. virens isolate (I10) have been UV mutagenised and selected for enzymatic activities related to mycoparasitic properties, such as cellulases, amylases, proteases, lipases. The ability of stable defective mutants to colonise S. sclerotiorum sclerotia is currently under evaluation by fluorescence microscopy. Biochemical studies have been planned in order to identify physiological functions potentially involved in sclerotia parasitization performed by the T. virens isolate under study. Since sclerotia are composed of an outer rind layer of melanized cells responsible for resistance to microbial degradation, assays for degrading activities specific for sclerotia phenolic components, such as lignin and melanin, are being performed on the I10 isolate. The molecular analysis aims to the identification of I10 genomic regions associated with the mycoparasitic ability. Based on both genetic and biochemical results, the identification of gene sequences related respectively to mutated functions or to physiological activities of interest is attempted. Initial experiments deal with ligninase and laccase genes, whose sequences are available in fungal databases. Such sequences have been exploited to design degenerated PCR primers in order to amplify, clone and sequence equivalent regions in T. virens.