Parasexual Crosses by Hyphal Anastomosis and Protoplast Fusion in the Entomopathogen Verticillium lecanii

SUMMARY: Sixty-two ‘crosses’ were attempted using mixed conidial suspensions and a ‘hanging drop’ technique involving 21 auxotrophic mutants obtained from seven isolates of Verticillium lecanil. Frequency of heterokaryon formation varied from 0 to 100%, with apparently complete incompatibility being expressed between some isolates, although this was influenced by the particular auxotrophic marker used. Protoplast fusion between compatible isolates usually resulted in prototrophic colonies, but was often unsuccessful when testing apparently incompatible isolates, suggesting that incompatibility was due mainly to nuclear or cytoplasmic factors, rather than to the wall. Evidence for the formation of prototrophic heterozygous diploids was provided by nutritional studies, and DNA content per conidiospore as measured by Feulgen microdensitometry. Conidiospore volume, determined by Coulter counter analysis, was not a reliable indicator of ploidy when investigating parasexual recombination between isolates of differing conidiospore size. Heterozygous diploids generally tended to be unstable, breaking down to yield recombinant haploids; however, four diploids were isolated which remained stable for 3-4 weeks on artificial media.