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

Abstract

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.

Analyses of air samples for ascospores ofLeptosphaeria maculans andL.biglobosa by light microscopy and molecular techniques

“…The technique of qPCR is increasingly being used in diagnostic estimation of spores and mycelia of fungal pathogens in the air and soil, as well as in infected plants (Walsh et al 2005;Fountaine et al 2007;Luo et al 2007). A cautionary report by McDevitt et al (2007) indicated that qPCR could be inhibited by masses of particulate matter of about 50 mg, and the amount of inhibition was positively correlated with the mass of particulate and the number of non-filamentous organisms.…”

Evaluation of eyespot incidence and structure of Oculimacula spp. population in winter rye in Lithuania

“…This method was used to quantify Oculimacula spp. in the eyespot-diseased winter rye stems (Walsh et al, 2005). In Lithuania, the ratio of O. yallundae to O. acuformis in the population has not been studied before.…”

Development of a real‐time PCR method for the identification of wheat genotypes carrying different eyespot resistance genes

“…2007), Fusarium graminaeum (Brandfass and Karlovsky 2006, Burlakoti et al. 2007), F. culmorum (Brandfass and Karlovsky 2006), OA and OY (Walsh et al. 2005).…”

“…Therefore, this method is not suitable for screening large segregating populations as needed in plant breeding. Moreover, the quantification of Walsh et al. (2005) is too expensive due to the high‐priced kits required and the use of TaqMan probes.…”