Rapid detection and diagnosis of Septoria tritici epidemics in wheat usinga polymerase chain reaction PicoGreen assay

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