Benomyl-Induced Somatic Segregation in DiploidArmillaria mellea

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Abstract
A. mellea is a bifactorially heterothallic fungus but, unlike other hymenomycetes, it has a persistently diploid vegetative phase in nature. Genetic analyses of A. mellea have been limited because this organism does not fruit reliably in culture. In order to induce somatic segregation of genetic markers, several diploid strains representing 4 of the intersterility groups or biological species of A. mellea in North America were treated with the fungicide benomyl. These diploids were heterozygous at the 2 mating-type loci and, in some cases, at nutritional loci as well. Somatic segregants of diploids were detected by testing 750 hyphal fragments derived from benomyl-treated material for auxotrophic phenotype and/or by visually selecting colonies that were morphologically different from the diploid progenitor. The mating types and auxotrophic requirements of each segregant were then determined. Although many of the segregants were homo- or hemizygous at each marker locus, some segregants were heterozygous at one locus or more, even when subcultured repeatedly in the absence of benomyl. Recombination was observed between all pairs of marker loci present in diploids synthesized in the laboratory. Because this protocol allowed recovery of numerous somatic segregants, parasexual genetic analysis in A. mellea can now proceed in the absence of in vitro fruiting.