1.8.21
INDUCTION AND ISOLATION OF TOXIN-DEFICIENT MUTANTS OF FUSARIUM SOLANI F.SP. GLYCINES

S LI1, GL HARTMAN1,2 and JW WIDHOLM1

1Department of Crop Sciences, and 2USDA-ARS, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

Background and objectives
Sudden death syndrome (SDS) of soybean (Glycine max) is caused by the soilborne fungus Fusarium solani f.sp. glycines. The most conspicuous symptoms of this disease [1] occur on leaves, although the fungus has been isolated only from roots and lower stems of plants. Foliar symptoms include interveinal chlorosis and premature defoliation. These symptoms were proposed to be caused by fungal toxins produced in infected roots and translocated to leaves. SDS is a relatively new disease and information about the genetics of this causal organism is not well documented. Identification and characterization of the fungal toxin(s) are still in progress. Generation of toxin-deficient mutants of the causal fungus may provide a useful tool to determine the importance of the toxin in SDS development in soybean. The objectives of this study were (i) to use chemical and physical mutagenesis to induce toxin-deficient mutants in F. solani f.sp. glycines; (ii) to evaluate the relationship between mutant types, such as morphological characters and cycloheximide resistance, and their toxin-producing ability; and (iii) to determine if the toxin is required for pathogenicity and SDS development.

Materials and methods
An SDS-causing isolate, Mont-1, was mutagenized by treating germinating macroconidia with ethyl methanesulphonate (EMS). In addition to chemical mutagenesis, both wild-type and the EMS-treated macroconidia were exposed to ultraviolet light (UV) to a lethal rate of at least 99%. Toxin-deficient mutants were identified by assaying their cell-free culture filtrate using a soybean seedling stem-cutting assay [2]. Cycloheximide-resistant mutants were isolated from potato-dextrose agar (PDA) containing cycloheximide at a concentration of 200 mg/ml, that is at least two times greater than the minimum inhibitory concentration (MIC) of the wild-type isolate. The resistance was confirmed by subculturing mutants on PDA for 1 month and retesting them on a cycloheximide-containing medium. Radial growth of mutants was assessed by measuring colony diameter after 10 days on PDA in dark at 23C. Macroconidial morphology and nuclear condition were examined with an Olympus BX 60 epifluorescence microscope at x400.

Results and conclusions
The wild-type Mont-1 isolate generally forms an appressed or semi-appressed colony with blue to blue-green pigmentation on PDA medium. Macroconidia were mostly sickle-shaped with four cells, but 3-, 5- and 6-celled macroconidia were occasionally found. After mutagenesis with EMS, 64% of the colonies differed from wild-type with nine different kinds of culture morphologies. Although UV survivors from wild-type macroconidia had more uniform colonies than EMS-survivors, 41% of the colonies had a blue centre surrounded by a white ring and had a blue ring at the outer colony margin. However, no correlation was found between colony morphology and toxin production. Stem-cutting assays indicated that morphological mutants tested retained their toxin-producing ability and caused SDS symptoms. One toxin-deficient mutant, Mu66, with a wild-type-like culture morphology, did not cause SDS symptoms in the stem-cutting assay. Another mutant, Mu119, caused delayed and very mild SDS symptoms. Culture morphology and radial growth of these two mutants were similar to that of the wild-type. In addition, 71 cycloheximide-resistant mutants were also obtained. Their culture filtrates are being tested using the stem-cutting assay to determine if they are toxin-deficient mutants. Further studies will be conducted to test mutant pathogenicity.

The mutants obtained in this study can be used as a genetic marker and may provide a useful tool for the investigation of the mechanism of SDS disease development and analysis of the relationship between toxin production and pathogenicity.

References
1. Hartman GL, Noel GR, Gray LE, 1995. Plant Disease 79, 314-318.
2. Li S, Hartman GL, Widholm JM, 1997. Phytopathology 87, S58.