| Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces multiple host-selective toxins (HSTs), including Ptr ToxA, encoded for by ToxA, Ptr ToxB, encoded for by ToxB , and Ptr ToxC. Variable distribution of these three HSTs among different isolates of P. tritici-repentis, both singularly and in all possible combinations, defines a complex race structure for this fungus. Eight races have been formally published. P. tritici-repentis race identification is typically based on inoculation of a standard set of host differentials. However, we found that a combination of phenotypic and genotypic characterization can be used to designate new races of P. tritici-repentis , and in some cases, is necessary to reveal variability not apparent by phenotype alone. In fact, genotypic and phenotypic characterization revealed two new races, which we designated race 9 and race 10. To aid in race classification, we utilized the association between HST production and race identity to develop a multiplex PCR assay based on HST gene-specific primers. Application of our multiplex PCR assay revealed that ToxB homologs, designated PbToxB, are present in P. bromi, causal agent of brownspot of bromegrass. Southern analysis of P. tritici-repentis and its Pleosporalean relatives anchored in a phylogram suggested that the distribution of ToxB extends to other plant pathogens in the Pyrenophora and Pleosporaceae. A search of available fungal genomes identified a distant homolog in Magnaporthe grisea , causal agent of rice blast. Due to the relatedness of P. tritici-repentis and P. bromi, and that of their grass hosts, we sought to determine if the ToxB homologs in P. bromi act as HSTs in brownspot of bromegrass. Though Pb ToxB does not act as an HST for P. bromi on tested bromegrass, evidence suggests PbToxB may function for P. bromi on tested wheat differentials. Alternatively, variation in functional homologs of ToxB may represent evolutionary remnants of an arms race between ascomycetes and their grass hosts. Several valuable tools were developed as a result of this work. In addition to multiplex PCR, we constructed a set of fluorescent protein expression vectors that we anticipate will be useful for a variety of applications in fungal-related research areas. |
