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Rapid detection and diagnosis of Septoria tritici epidemics in wheat using a polymerase chain reaction/PicoGreen assay

In order to detect and quantify Septoria tritici infection levels in wheat leaves, a polymerase chain reaction (PCR) assay was developed using the β‐tubulin gene as target. Specific PCR primers were designed by aligning and comparing β‐tubulin sequences from other fungi. The final primer set was selected after being tested against several fungi, and against S. tritici‐infected and uninfected wheat leaves from different localities. A single DNA fragment (496 bp) was amplified from S. tritici, whereas no products were generated from DNA of the host plant or other micro‐organisms associated with wheat leaves. Using agarose gel analysis, approximately 2 pg S. tritici genomic DNA could be detected in each assay. However, for rapid quantification of PCR‐amplified products, a fluorometric microtitre plate‐formatted PicoGreen assay was used; this could detect as little as 10 pg S. tritici DNA in the presence of 200 ng wheat leaf DNA. The PCR/PicoGreen assay was applied successfully to study the colonization, infection and subsequent disease development of S. tritici on wheat, both under controlled conditions in the glasshouse and in the field.