Project Number: 5442-21220-023-53
Project Type: Specific Cooperative Agreement

Start Date: Jun 01, 2008
End Date: May 31, 2009

Objective:
The long-term goal of this project is to develop effective, long-term, and environmentally sound disease control methods for Sclerotinia diseases in a broad range of corps. This goal is being pursued through basic molecular genetic and biological studies of S. sclerotiorum that critically test hypotheses related to the mechanisms of Sclerotinia pathogenesis. In line with this goal, the specific objective of this project is to determine the roles and requirements for oxalic acid in the disease process on sunflower, dry bean, canola, and pulse crops. Oxalic acid has been implicated as an essential pathogenicity factor or as an important virulence factor in a number of independent reports. These studies have ranged from physiological studies correlating exogenous oxalic acid treatment with symptom development, to inoculation studies using natural isolates or mutants of S. sclerotiorum that produce little or no oxalic acid. The correlations made from these studies have led to the popularly implemented strategy of transgenically engineering hosts to express oxalate oxidase or oxalate decarboxylase activities. These efforts have resulted in promising preliminary reports of partial or complete resistance to Sclerotinia. Yet despite the millions of private and public dollars that have been invested in these strategies, no large-scale field trials have validated this approach, and resistant cultivars have not materialized. Likewise, plant screens to identify genotypes with increased tolerance to oxalic acid have not supported a correlation between oxalate tolerance and disease resistance. Silver bullets are desirable but rare. If we are to control Sclerotinia diseases we must understand the pathogen. We must understand the specific roles of various pathogen-produced factors using systematic, genetically defined and robust methods. As such, the specific objective of this proposal is to use a genetically-defined mutant in the oxalate biosynthetic pathway to determine the full range of phenotypic consequences associated with the inability to produce oxalic acid during plant infection. We recently created this mutant in my research program and found that it produces no detectable oxalic acid under any tested condition in vitro or in planta. Despite this complete lack of oxalic acid production, this mutant is still capable of infecting plants and causing disease symptoms in some interactions. Under what conditions does disease still occur? Is there variation among hosts regarding the requirement for oxalic acid? To address these questions, we will investigate these mutants 1) microscopically for the ability and mechanism used to penetrate leaf and stem tissues of the above mentioned crops; 2) to determine if the nutritional status of inoculum has an effect on the ability to cause disease; and 3) determine if the physiological status of the host has an effect on susceptibility.

Approach:
The approach implemented here begins with the use of a genetically-defined mutant of S. sclerotiorum that does not produce oxalate as a result of a mutation within the oxalate biosynthetic pathway. Studies in the past have relied on natural isolates or selected strains that varied in oxalate production. A disadvantage of working with such strains is that one does not know the genetic basis of the phenotype and consequently multiple mutations or pleotropic effects may exist. The mutant used in this study is a gene deletion strain specifically defective in its ability to produce oxaloacetate acetyl hydrolase. This mutation results in a specific and complete loss of ability to synthesize and secrete oxalic acid. Our observations with two independent mutants for this gene suggest that it retains pathogenicity in some interactions although virulence and symptom expression are attenuated. The experiments proposed here will test the hypothesis that oxalic acid is not an essential pathogenicity factor for Sclerotinia diseases. We will inoculate a diverse set of hosts including sunflower, dry beans, canola, chickpea and lentils to determine if oxalate is required for infection of some hosts but not others. These infection studies will be followed by microscopic observations to determine how host penetration and colonization differs in oxalate producing and non-producing strains and how the nutrient status of the infecting hypahe alters the infection capacity. Finally, we will examine varying host tissues (roots, stems, flowers and leaves) and various ages to determine if host physiology affects susceptibility to oxalate non-producing strains. These studies will provide a clearer understanding of when and where oxalate is needed for disease development and will guide more rational approaches for disease management through the manipulation of oxalate accumulation during infection.