Characterization of the parasexual cycle in the eyespot fungus, Pseudocercosporella herpotrichoides

Abstract

Parasexual recombination in the eyespot fungus, Pseudocercosporella herpotrichoides , is demonstrated using complementary auxotrophic mutants derived from the W-type isolate 22–20. The parasexual cycle follows a typical pattern consisting of a mosaic heterokaryon phase, a relatively stable diploid phase and various putative aneuploid stages leading to recombinant haploid formation. The heterokaryon can be resolved into its component homokaryons either by isolating single hyphal tips or from single conidia, which remain homokaryotic. The non-sporing diploids can be isolated from random plating of spores and hyphal fragments taken from heterokaryotic colonies, or by regenerating protoplasts isolated from heterokaryotic mycelia. An efficient method for obtaining diploids was developed, which exploits the particularly slow growth rate of heterokaryons formed between complementary histidine auxotrophs ( Hisl, Hien, Hi ). Diploid mycelium grows out as faster growing sectors from such heterokaryons. Segregation yielding recombinant haploids followed exposure to 5-fluorouracil. Various putative aneuploid stages were observed, typified by their very slow growth rate and instability. Spontaneous outgrowths yielding stable recombinant types were recovered from these unstable colonies following maceration of mycelium and plating on a non-selective, complete medium. A cross was performed to investigate the inheritance of benzimidazole and N -phenylcarbamate fungicide resistance. After retesting of resistance phenotypes, cosegregation of carbendazim resistance and increased sensitivity to the phenylcarbamate MDPC was confirmed in most cases. One exception involved a putative recombinant in which sensitivity to both carbendazim and the phenylcarbamate was combined. This recombinant was obtained after exposure of the original diploid to 5-fluorouracil, known to stimulate mitotic crossing-over in other fungi. However the possibility remains that it represents a chance spontaneous mutation either in the resistance gene or at some other locus.