PCR-based assays to assess wheat varietal resistance to blotch (Septoria tritici and Stagonospora nodorum) and rust (Puccinia striiformis and Puccinia recondita) diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect a nd quantify four fungal foliar pathogens in wheat. For Septoria tritici (le af blotch) and Stagonospora nodorum (leaf and glume blotch), the beta -tubu lin gene was used as the target region. Diagnostic targets for Puccinia str iiformis (stripe or yellow rust) and P. recondita (brown rust) were obtaine d from PCR products amplified with beta -tubulin primer sequences. Final pr imer sets were designed and selected after being tested against several fun gi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultan eously, whereas no products were generated from wheat DNA or other non-targ et fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent d ye SYBR Green I, PCR-amplified products could be quantified individually, b y reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag l eaf of 19 cultivars at Growth Stage (GS) 60-64 in both 1998 and 1999. The i nfection levels for the cultivars were ranked, and showed, with a few excep tions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre- symptomatic infection levels was possible. Although sampling and DNA detect ion methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening t o measure the interaction between different pathogens and their hosts at di fferent growth stages, and in specific tissues. This should enable an earli er identification of specific resistance mechanisms in both early-stage bre eding material and field trials.