Overcoming vegetative incompatibility within and between pathotypes of Pseudocercosporella herpotrichoides by protoplast fusion

Abstract

Vegetative incompatibility between isolates of Pseudocercosporella herpotrichoides was detected using complementation testing on minimal medium between auxotrophic mutants obtained following uv mutagenesis of conidia. This analysis confirmed earlier data indicating the occurrence of vegetative incompatibility in this fungus. All combinations of W-type with R-type isolates were incompatible. Incompatibility was also shown between the three W-type isolates tested. Three of the four R-type isolates showed full mutual compatibility, while a fourth isolate was fully compatible with only one of these strains. This latter isolate exhibited partial compatibility to a second isolate, depending on the auxotrophic phenotype of the strains tested, and was incompatible with the third isolate in all strain combinations. Using the protoplast fusion technique it was possible to overcome vegetative incompatibility in all pathotype combinations: W × W, R × R and W × R. Fusion frequency in incompatible W × W and W × R crosses was typically ten fold lower than in crosses between vegetatively compatible strains. Fusion of incompatible R-type isolates was obtained at a similar frequency to compatible strain combinations. Fusion products were not heterokaryotic, since isolated, uninucleate protoplasts were able to regenerate on minimal medium. Regeneration of protoplasts on diagnostic media containing fungicides confirmed the presence of both parental genotypes in several of the crosses. The use of fungicide resistance in combination with auxotrophic requirements allowed the selection of fusion products in crosses with fungicide sensitive wild-type isolates.