Gaeumannomyces graminis vars. avenae, graminis, and tritici identified using PCR amplification of avenacinase-like genes

Identifying take-all pathogens, Gaeumannomyces graminis varieties avenae (Gga), graminis (Ggg), and tritici (Ggt), is difficult. Rapid identification is important for development of disease thresholds. We developed a single-tube, polymerase chain reaction (PCR) method differentiatng among Gga, Ggg, and Ggt. Nucleotide base sequence analyses of avenacinase-like genes from Gga, Ggg, and Ggt isolates provided the basis for designing variety-specific primers, Sequences from Ggg and Ggt were highly related (99% identity), but Gga sequences were <95% identical to Ggg and Ggt sequences. Three 5' primers specific for Gga, Ggt, and Ggg and a single 3' common primer allowed amplification of variety-specific fragments of 617, 870, and 1,086 bp, respectively. Each 5' primer was specific in mixed populations of primers and templates. No PCR products were amplified from related fungi including Gaeumannomyces cylindrosporus and Phialophora spp. We surveyed 16 putative Ggt isolates using our assay; nine produced Ggt-specific fragments and seven produced Ggg-specific fragments. Five Gga isolates produced Gga-specific fragments. However, Gga- and Ggt-specific fragments were observed from a sixth Gga isolate, RB-W, which indicates a mixed culture or a heterokaryon. Our single-tube, PCR method rapidly differentiates among the important take-all pathogens commonly encountered together in cereal fields.