MOLECULAR APPROACHES TO IMPROVE THE EFFICACY OF FUNGAL BIOCONTROL AGENTS
Dipartimento di Arboricoltura, Botanica e Patologia Vegetale - sezione Patologia Vegetale, Universita degli Studi di Napoli "Federico II", via Universita 100, 80055 Portici (NA) Italy.
About 30% of the yield in agriculture is lost because of the combined effects of biotic and abiotic stresses, with pathogenic fungi alone responsible for a reduction of about 12%. Control of fungal pathogens is based on the use of agronomic practices and pesticides, but widespread application of chemicals inundates the agroecosystems with toxic compounds that affect the balance of the natural food chain. In addition, resistant and more virulent pathogen populations are selected causing an escalation in the amount of pesticides used. A variety of new technologies are being developed to integrate or substitute the application of chemicals in the attempt to reduce both the ecological and financial cost of disease control. Antagonistic microorganisms are being studied in depth and considered as an attractive option for the development of microbial-based biofungicides and mixed formulations containing biocontrol agent(s) and low doses of chemical fungicides. Successful and consistent results have been achieved with some biocontrol agents such as Agrobacterium and Bacillus, whose mechanism of biocontrol are largely understood. However, limitations in the practical use of bacterial agents often arose from the production of toxic substances and formulations with a short shelf-life. The application of fungal biocontrol agents has also been delayed because of difficulties in obtaining consistent results in biocontrol and the relatively poor understanding of the plant-microbe and microbe-microbe interactions involved in the antagonistic processes.
The most studied fungal biocontrol agents are Trichoderma spp. and some isolates, effective as biofungicides in certain culture conditions, have been recently introduced in commercial agriculture. Concurrently, fundamental discoveries concerning the mechanism of action of these fungi have been made. The involvement of mycoparasitism in biocontrol, and the role of synergistic mixtures of lytic enzymes andlor antibiotics, have been studied at the molecular level. For instance, research with disrupted mutants has recently indicated that more than a single component of the chitinolytic complex need to be impaired in order to substantially affect biocontrol. Studies on the mechanism of biocontrol have also indicated that Trichoderma and other mycoparasites have developed a vast array of molecular tools to support their parasitic behaviour. Genes encoding for cell wall degrading enzymes (CWDES) such as chitinolytic, glucanolytic and proteolytic enzymes have been isolated and used to improve biocontrol capabilities of Trichoderma strains. By increasing the number of gene copies or expressing the relevant genes under the control of strong constitutive or inducible promoters, biocontrol agents more lytic and potentially more effective than wild types have been obtained. This suggests the use of synergistic combinations of genes encoding for CWDEs to improve useful strains. The same genes have also been successfully introduced in bacterial agents to extend biocontrol abilities and in several crop plants to improve disease resistance. New possibilities come from recent studies of Trichoderma promoters involved in the expression of biocontrol-related genes and from the analysis of the early phases of mycoparasitism. Non-coding sequences activated during mycoparasitism have been characterized and are being used as biocontrol-inducible promoters to express in Trichoderma heterologous antifungal or reporter genes for biocontrol improvement and strain monitoring. Molecular studies on the mechanism of host-parasite recognition and on the early induction of mycoparasitism provide insight on how to modify these processes in order to enlarge the range of susceptible hosts and biocontrol applications. New reporter genes (i.e. the green fluorescent protein) are being used to identify molecules that activate the mycoparasitic cascade, which may permit the enhancement of biocontrol in the field without the use of genetically modified strains. Substantial work must still be done to achieve a complete understanding of the antagonistic mechanisms of fungal biocontrol agents, which reveal an astonishing level of complexity.