• biocontrol agent
• gene targeting
• homologous recombination
• mycoparasitism
• Trichoderma

The antagonistic properties of the genus Trichoderma make it an ideal candidate for biocontrol against phytopathogenic fungi in integrated pest management. Strains of Trichoderma spp. (including the T. virens I10 isolate) have proven to be effective mycoparasites of important plant-pathogenic fungi.
The mycoparasitic activity of Trichoderma spp. against sclerotial phytopathogenic fungi is a powerful tool for a biocontrol agent, since sclerotia are highly resistant structures representing the pathogen’s primary survival form in soil.
Parasitic interactions established by Trichoderma against sclerotial fungi have been mainly investigated by histological and biochemical analysis. Several enzymatic activities have been related to sclerotia mycoparasitism, such as enzymes degrading cell wall components (chitinase and cellulase) or phenolic compounds (lignin and melanin degrading enzymes), protease and lipase.
The aim of this work was to explore different enzymatic activities involved in the mycoparasitism deployed by T. virens against sclerotia of some phytopathogenic fungi.
The approach used for this study implicated an initial phase consisting in the production of random mutants and a next step of gene targeting mutagenesis.
UV random mutants were produced from the I10 isolate and two strains previously transformed with genes encoding for fluorescent proteins, I10 GFP and I10 DsRed. These mutants were screened for specific functions (cellulases, lipases, proteases and laccases) in order to evaluate their mycoparasitic ability against sclerotia derived by Sclerotinia sclerotiorum and Botrytis cinerea. Data obtained from 10 stable UV mutants challenged with sclerotia showed in some cases a correlation between the putative mutation and mycoparasitic activity, although a different behaviour versus the two pathogens was observed.
Gene targeting by homologous recombination (HR) is a powerful technique that can be applied to study gene functions and to alter interesting properties of fungal strains. However, in filamentous fungi this is often hampered by very low frequencies of HR and it is therefore not trivial to target a gene of interest to a specific genomic locus or to delete an endogenous gene. To increase the recombination rate in T. virens transformation the orthologue of the human KU70, required for the nonhomologous and joining (NHEJ) pathway and responsible for ectopic DNA integration, has been identified and deleted. The effect on gene targeting of the absent ku70 in T. virens was tested by deleting a laccase gene. Efficiency of gene targeting was 90% in the I10 Δku70 strain, which is a significant increase compared to the parental strain (non-ku70 deleted) where only a 10-15% gene knock-out frequency was observed. The double mutant T. virens Δku70/Δlac was used to study the potential role of the laccase gene product in mycoparasitism against sclerotial fungi, such as B. cinerea or S. sclerotiorum.
The ability of T. virens Δku70/Δlac to decay sclerotia was evaluated and again it came out to be different against the two pathogens. The mycoparasitic activity was surprisingly higher in the interaction with S. sclerotiorum sclerotia while it was as expected significantly impaired vs B. cinerea sclerotia.
Data presented in this thesis have provided several outputs related to the mycoparasitic interaction between T. virens and S. sclerotiorum or B. cinerea. A new gene function (laccase) has been analysed in T. virens: six genes have been identified and one of them has been knocked out and partially characterized in the biological system under study.
New investigations on the laccase gene family in T. virens could be attractive, being those enzymes useful biocatalysts for several biotechnological applications.
Finally the Δku70 strain represents a powerful tool for a high-throughput functional gene analysis in a T. virens strain proven to be an efficient biocontrol agent.