Selection and Culture of Auxotrophic and Drug-Resistant Mutants of Tilletia caries

Churchill, A. C. L., and Mills, D. 1984. Selection and culture of auxotrophic and drug-resistant mutants of Tilletia caries. Phytopathology 74:354-357. Mutagenesis and selection procedures for isolation of auxotrophic in the presence of cycloheximide. Paired, sexually compatible, auxotrophic, mutants of 7Tlletia caries race T-1 were developed. Mutants with haploid strains produced mycelial growth on minimal medium, presumably requirements for adenine, uracil, glycine, or a general nitrogen source were because of heterokaryosis. Secondary sporidia derived from these matings identified. Only spontaneous mutation to cycloheximide resistance was exhibited the nutrient requirement of either parent, indicating that the observed in tests with 12 growth-inhibiting drugs. These latter mutants had frequency of reverse mutation and stable diploid formation was low. restricted colony growth and generally failed to produce secondary sporidia Additional key words: common bunt, complementation, smut. The bunt fungi of the genus Tilletia are among the most and lunate secondary sporidia were produced for mutagenesis destructive cereal pathogens (7); however, studies concerning the experiments in liquid T-19 medium (27), referred to in this report as genetics of pathogenicity of these smut fungi have not kept pace medium A. The cultures were maintained by transfer of an aliquot with similar studies of other plant pathogens (8). The slow progress (0.5 ml) of inoculum to 50 ml medium A at 5to 7-day intervals. The in understanding the genetics of the common wheat bunt pathogen, generation time for each strain has been described previously (3). Tilletia caries (DC.) Tul., and related fungi can be attributed in part Two additional media were used to isolate and characterize to the absence of experimental strains with suitable genetic auxotrophic strains. A minimal medium, medium B, differed from markers, medium A in that KNO 3 (4 g/L) was substituted for asparagine, Previous interspecific and intraspecific hybridization studies and Noble agar (1.5%) was used as a solidifying agent when have examined the inheritance of diploid parental traits of Tilletia required. A complete medium, medium C, contained the spp. (6,11-13,23,24). These studies used properties such as constituents of medium A plus nucleic acid bases (10 mg/L), pathogenicity, teliospore morphology, teliospore germination, and Edamin amino acids (1 g/ L) (Sheffield Chemical Co., Norwich, NY sorus shape as characters for segregation analysis. Expression of 13815), and an aliquot (10 ml/L) of 100 X vitamin solution. The these factors was highly variable, and progeny often showed a vitamin solution contained riboflavin, pyridoxine-HCl, and pcontinuous range of characteristics rather than discrete parental aminobenzoic acid (each 50 mg/ L); calcium pantothenate, phenotypes. Genetic studies of other plant-pathogenic fungi have nicotinic acid, and choline chloride (each 200 mg/ L); inositol (400 commonly used biochemical mutations controlled by single genes mg/L); and biotin (2.6 mg/L). All cultures were incubated in the as nuclear genetic markers, in addition to general physiological or dark at 19 C. Liquid cultures were grown on a rotary shaker (175 morphological traits (1,4,10,15,18,22). Similar mutants of T. caries rpm). and related bunt fungi would greatly facilitate genetic studies of Mutagenesis and selection of auxotrophs. Log-phase 5to 7-day these pathogens. old liquid cultures of secondary sporidia were harvested from This paper reports the development of mutagenesis and selection medium A for mutagenesis experiments by filtration through procedures for isolation of biochemical and drug-resistant mutants sterile 20 -Am nylon mesh. The concentration of sporidia in the of T. caries. Sexually compatible auxotrophic strains were used in filtrate was estimated with a Neubauer hemacytometer, and the complementation tests, and the resulting prototrophic growth was cells were collected by centrifugation at 6,000 g for 10 min., then examined for heterokaryosis. T. caries was selected for use in this washed and resuspended in medium A. Sporidia (2 X 107 cells per investigation over other wheat bunt fungi because of the milliliter) were exposed to 100 lzg/ml N-methyl-N'-nitro-Navailability of monosporidial haploid strains with favorable nitrosoguanidine (NTG) (Aldrich Chemical Co., Milwaukee, WI laboratory culture characteristics. 53233) in 0.2 M acetate buffer, pH 5.6, for 20 min to 3 hr on a rotary shaker. Aftgr the exposure period, the NTG suspension was diluted MATERIALS AND METHODS with 15 volumes of medium C, and the sporidia were collected by centrifugation and washed. They were then transferred to 50 ml Strains and media. Monosporidial haploid strains of T. caries liquid medium C in flasks and incubated on a rotary shaker for 5 (race T-l) no. 18 (+ mating type) and nos. 24 and 26 (mating type) days to permit the cells to recover from the mutagen treatment (14) were maintained on noncommercial potato-sucrose agar (17,26). Secondary sporidia from these cultures were harvested (PSA) at 4 C and subcultured at 2-mo intervals. Abundant filiform by filtration, washed twice in medium B, and either transferred directly to medium C plates (total isolation procedure) or subjected The publication costs of this article were defrayed in part by page charge payment. This to filtration enrichment (29). article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § Filtration enrichment of mutant cells consisted of two 1734 solely to Indicate this fact. consecutive incubation periods in medium B, which prohibited 01984 The American Phytopathological Society growth of auxotrophs. Ungerminated sporidia, including putative