W KOELLER

Cornell University, Department of Plant Pathology, New York State Agricultural Experiment Station, Geneva, NY 14458, USA

The best understood fungal organisms Saccharomyces cerevisiae, Aspergillus nidulans and Neurospora crassa are saprophytes lacking traits of pathogenicity. The recent cloning and sequencing of the entire S. cerevisiae genome in combination with tools provided by genomics and biomformatics offers an opportunity to evaluate the question, whether mode of fungicide actions identified for S. cerevisiae can be easily transferred to plant pathogens. The new class of strobilurin fungicides will serve as an example. Strobilurins have been introduced as broad-spectrum agricultural fungicides, and their mode of action was identified as the inhibition of respiration by binding to cytochrome b. The initial mode of action studies were conducted with liver mitochondria, and respective results were confirmed with yeast mitochondria and then with mitochondria isolated from plant pathogens such as Pyricularia oryzae and Septoria tritici. However, our own work with Venturia inaequalis and results provided for other pathogens clearly indicated that the primary strobilurin mode of action is not sufficient to explain the inhibition of fungal growth. The sometimes striking discrepancies between inhibitory in Vitro and in vivo effects of strobilurins was explained by the interference of alternative respiration with strobilurin action. Active oxygen radicals formed in the presence of a cytochrome b blocked by a strobilurin induce the expression of alternative oxidase. The protective enzyme is imported into mitochondria and circumvents the strobilurin inhibitor site. This circumvention mechanism is not active in S. cerevisiae, because this highly specialized fermentative yeast apparently lacks an alternative oxidase gene. The example illustrates that the primary mode of action of a fungicide can be determined with S. cerevisiae. It also suggests that nonpathogenic organisms such as S. cerevisiae can be of limited value in studies aimed at the understanding of modes of fungal growth inhibition, because metabolic events in addition to the inhibition of a primary target site are determinants of a more comprehensive scenario of inhibitor activities.

The recent revolutions in several disciplines such as combinatorial chemistry, automated high-throughput screens and genomics will have profound impact on the process of discovering new bioactive compounds including agricultural fungicides. The new discovery route starts with defining desired inhibitor targets, proceeds to the validation of such targets and then establishes screening systems suitable for identifying specific inhibitors of such targets. In this scheme of fungicide discovery, the mode of action of a perspective fungicide is predetermined. The most important step in this process is the validation of the target site chosen, because it determines the design and ultimate success of a particular screening system. Fungal cutinases will serve as an example for the restrictions but also opportunities inherent to this discovery approach. The plant cuticle and in particular the polyester cutin comprises the first barrier that has to be overcome by fungal pathogens invading their host plants. For more than two decades, experimental evidence had suggested that cutinases produced by plant pathogens thriving on cutin as a saprophytic carbon source are also involved in overcoming the cuticle barrier during infection. The technique of specific gene disruption left no doubt that this model was too simplistic. For example, Fusadum solani and Alternaria brassicicola cutinases expressed during stages of saprophytic digestion of cutin were very similar. Disruption of respective genes, however, had no impact on the pathogenicity of such gene-disrupted mutants. The discovery that the nonpathogenic fungus Aspergillus oryzae produced a lipase very similar to cutinases expressed by plant pathogens during saprophytic conditions strengthened the hypothesis that such cutinases with functions of a 'multi-purpose' lipase reflect saprophytic rather than pathogenic traits of fungi. However, our work with A. brassicicola suggested also that a different class of cutinases with potential functions in host invasion exists in plant pathogens. The cutinase example illustrates that predetermined modes of actions of future fungicides must not only focus on plant pathogens; they also must differentiate between saprophytic and pathogenic stages of these pathogens.