Jarod Schweighart, Toumas Hatinen, Norbert C. Furumo and Steven L. Daniel
Departments of Chemistry and Biological Sciences, Eastern Illinois University, Charleston, IL

Sclerotinia stem rot is a serious yield-reducing soybean disease caused by the fungal pathogen Sclerotinia sclerotiorum. The ability of this fungus to infect soybeans and other crops appears to hinge on its ability to produce oxalate. Presently, little is known about the synthesis of oxalate by this organism. The goal of this project was to determine the impact that carbon compounds have on growth and oxalate production by this fungal pathogen. S. sclerotiorum Arg-L was grown at 25^oC with shaking in an undefined medium (minerals, 0.1% yeast extract) containing 20 mM glucose and one of the following co-substrates (20 mM): acetate, malate, succinate, glyoxylate, pyruvate, or glycolate. Co-substrate concentrations were monitored by HPLC while glucose was determined using an enzyme assay. Growth (dry weight of mycelia) was significantly stimulated by the presence of malate or succinate whereas glycolate and pyruvate slightly repressed growth. Oxalate production was greatest with malate followed by succinate or acetate as co-substrates. Glycolate and pyruvate repressed oxalate synthesis. These results suggest that carbon sources regulate growth and oxalate synthesis by S. sclerotiorum and that nutritional factors may impact the virulence of this fungal pathogen.

Introduction

Sclerotinia sclerotiorum is a pathogenic fungus that causes Sclerotinia stem rot (SSR) of soybeans (Figure 1). While this pathogen has been implicated in plant disease for over 40 years, it has only become a problem for soybean farmers since the early 1990s with outbreaks becoming more common and widespread (1). Worldwide, SSR reduces soybean yields by more than 1 million metric tons a year. In the United States, SSR reduces soybean yields by nearly half a million metric tons a year, costing farmers ~100 million dollars. This pathogen also infects over 400 plant species, including vegetable, ornamental, fruit, and weed species. How S. sclerotiorum infects plants and causes disease is complex and not well understood. However, it is known that S. sclerotiorum produces large amounts of oxalate during infection and that oxalate is a pathogenicity determinant. Strains of S. sclerotiorum which do not produce oxalate do not infect plants (2). While many studies have been done to address the role of oxalate in the disease process, little is known about the mechanism by which S. sclerotiorum synthesizes oxalate. The goal of this project was to determine how carbon source impacts growth and oxalate biosynthesis by S. sclerotiorum.

Materials and Methods

Culture Conditions. Sclerotinia sclerotiorum strains Arg-L and DE-7 were grown at 25oC with shaking (300 rpm) in culture flasks containing an undefined culture medium. The culture medium contained 0.1% yeast extract and was supplemented with no substrates (control), 20 mM glucose alone, or 20 mM glucose plus 20 mM acetate, glycolate, pyruvate, succinate, malate, or glyoxylate as co-substrates.  

Biomass (Growth) Measurements. Biomass (fungal mycelium) was collected from culture flasks on membrane filters and dried at 60^oC for 72 hours, cooled, and weighed to determine the milligrams (dry weight) of mycelium formed during incubation.

Analytical Techniques. Concentrations of oxalate and co-substrates in culture filtrates were determined using a Beckman HPLC and a 300-mm BioRad Aminex HPX-87H column. HPLC conditions were: column temperature, 55^oC; flow rate, 0.6 ml/min of 0.01 N H₂SO₄; injection volume, 10 µl; and detector, 210 nm. Glucose concentrations were determined using a Sigma Glucose Diagnostic Kit.

Results

Based on oxalate-to-biomass ratios, strains Arg-L and DE-7 displayed differential responses to the various co-substrates (acetate, glycolate, pyruvate, succinate, malate, and glyoxylate):

1. With Arg-L, acetate, malate, and glyoxylate as co-substrates yielded the most oxalate per unit of biomass formed (Table 1).

2. With DE-7, acetate, pyruvate, succinate, and malate as co-substrates yielded the most oxalate per unit of biomass formed (Table 2).

3. With both organisms, glycolate as a co-substrate yielded the least amount of oxalate per unit of biomass formed.

4. With strain Arg-L, none of the co-substrates were growth supportive when glucose was omitted from the medium.

Controls and glucose-supplemented cultures produced essentially equal amounts of oxalate (Figs. 2 and 3).  In all cultures, glucose was completely consumed by day 9 of incubation (Figs. 3, 4, 5, 6, 7, 8 and 9). Acetate, succinate, malate, and pyruvate were utilized simultaneously with glucose consumption (Figs. 4, 5, 6 and 7).  Negligible amounts of glyoxylate and glycolate were consumed during growth (Figs. 8 and 9).  Glycolate as a co-substrate repressed oxalate synthesis (Fig. 9).  Of the substrates tested, glucose cultures supplemented with acetate, succinate, or malate yielded the most oxalate (Figs 4, 5, 6 and 7).

Figure 2.	Figure 3.	Figure 4.	Figure 5.
Figure 6.	Figure 7.	Figure 8.	Figure 9.

Growth and substrate/oxalate profiles indicate that carbon source influences oxalate biosynthesis in S. sclerotiorum.  Acetate, succinate, and malate lead to significant oxalate production probably via the TCA cycle and the synthesis of oxaloacetate.  Oxaloacetate is converted to acetate and oxalate by oxaloacetate hydrolyase, an enzyme found in the oxalate-producting fungus Aspergillus niger (2). However, we have been unable to detect this enzyme in S. sclerotiorum.  Glyoxylate did not significantly stimulate oxalate production, suggesting glyoxylate dehydrogenase (an enzyme found in the oxalate-producing, fungal pathogen Sclerotium rolfsii [2]) is not involved in oxalate synthesis in S. sclerotiorum. We have also not detected this enzyme in S. sclerotiorum.  Studies are currently underway to detect enzymes involved in oxalate biosynthesis in S. sclerotiorum (Fig. 10).

[1] Compendium of Soybean Diseases. 1999. Fourth edition (editors: G. L. Hartman, J. B. Sinclair, and J.C. Ruppe). The American Phytopathological Society.

[2] Dutton, M. V., and Evans, C. S. 1996. Oxalate production by fungi: its role in pathogenicity and ecology in the soil environment. Can. J. Microbiol. 42:881-895.