Interspecific hybridisation between Pseudocercosporella herpotrichoides and P. anguioides achieved through protoplast fusion

Abstract

The relatedness of the cereal eyespot pathogen, Pseudocercosporella herpotrichoides , to the weakly pathogenic species P. anguioides was investigated using protoplast fusion to produce somatic hybrids. Crosses between P. anguioides and strains representing the two main pathotypes of P. herpotrichoides (W and R) were conducted. Procedures for protoplast isolation and regeneration developed for P. herpotrichoides were successfully used, without modification, with P. anguioides . PEG-induced fusion of protoplasts, isolated from auxotrophic mutants of P. herpotrichoides and P. anguioides , generated prototrophic fusion products on minimal regeneration medium at low frequency (0·00016%–0·0016%). Subculture of fusion products on non-selective media generated spontaneous segregants with a range of morphological phenotypes. Characterization of parental nutritional and isozyme markers revealed novel marker combinations, confirming that genetic recombination between the species had occurred, presumably by parasexual processes following karyogamy. Both pathotypes of P. herpotrichoides appeared able to hybridize with P. anguioides under these conditions. Marker segregation was not random, suggesting structural differences in the genomes of the two species of the expression of genomic incompatibility. Determination of spore sizes of species and hybrids showed that the recombinants formed conidia intermediate in length between the parental species. Mean cell length of conidia remained relatively constant at 9·7±0·2 m (standard error) per cell, with longer spores containing more cell compartments. No evidence to indicate that the hybrid recombinants were diploid was obtained from the spore analysis and these strains remained stable after exposure to the haplodizing agent carbendazim. Clear evidence for the segregation of pathogenicity to wheat cv. Beaver was obtained from a 9 wk pathogenicity test with a range of hybrids and parental isolates. P. anguioides was only weakly pathogenic to wheat, whereas both pathotypes of P. herpotrichoides caused severe lesion development. Interspecific hybrids varied in pathogenicity. Some were non-pathogenic, like the P. anguioides parent, while others gave infection levels comparable to the P. herpotrichoides R-type parent. Pathogenicity was not impaired by the presence of the cysteine auxotrophic marker of the R-type. The successful recovery of interspecific hybrids indicates that these two species are relatively closely related.