Mutations in Ophiostoma ulmi induced by N-methyl-N′-nitro-N-nitrosoguanidine

Abstract

Mutations were induced in Ophiostoma ulmi, the causal agent of Dutch elm disease, by treating yeastlike cells of wild-type strains with the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The induced frequency of mutation, assessed by scoring the frequency of benomyl-resistant mutants among the surviving population, was highest in treatments that left from 4 to 10% survivors. The survival rate of the cells was affected by mutagen concentration and length of exposure to MNNG, as well as by cell concentration, cell age, pH, and chemical composition of the incubation buffer. Optimal conditions for routine induction of mutants were obtained by resuspending exponentially growing cultures in phosphate buffer at pH 7.5 (2.0 × 10⁷ cells/mL) and treating the cells with MNNG (20 μg/mL) for 90 min with agitation. The proportion of auxotrophs among the survivors increased at least 200-fold when mutagenesis was followed by nystatin enrichment. Most auxotrophs tested were sexually fertile and carried a recessive mutation at a single nuclear locus. The benomyl-resistant phenotype was dominant. Key words: Ophiostoma ulmi, Dutch elm disease, mutations, N-methyl- N′-nitro-N-nitrosoguanidine.