Using Real-time PCR to Discriminate and Quantify the Closely Related Wheat Pathogens Oculimacula yallundae and Oculimacula acuformis

Oculimacula yallundae and O. acuformis are the causal agents of eyespot disease of wheat and other cereals. The two fungi react differently to the application of fungicides, but they cannot be distinguished visually and their occurrence is often masked by other less damaging pathogens. Current methods to detect and distinguish Oculimacula species are impractical when testing large numbers of samples. A real-time polymerase chain reaction (PCR) assay, suitable for largescale testing, was developed and used for quantitative detection and discrimination of O. yallundae and O. acuformis. As the available DNA sequences differ by only a small number of conserved nucleotide polymorphisms, three different methods were investigated to achieve the desired specificity. A combination of mutagenically separated PCR and a shortened primer gave the best specificity for O. yallundae and O. acuformis, respectively. Although the comparison illustrated the practicalities of each method, it was not possible to devise a fixed set of rules for the design of primers to discriminate two closely related sequences; it is assumed that sequence context is an overriding and difficult to predict factor, and that a range of empirical approaches may need to be taken to reach the specificity required