Submitted to: Phytopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/21/2006
Publication Date: 6/1/2007
Citation: Barnes, C.W., Szabo, L.J. 2007. Detection and identification of four common rust pathogens of cereals and grasses using real-time polymerase chain reaction. Phytopathology. 97:717-727. Interpretive Summary: Rust fungi are widespread pathogens of cereals, such as wheat, that annually cause significant yield losses worldwide. However, these fungi are often difficult to identify because of similar spore and symptom characteristics, especially during disease development. Therefore, real-time PCR assays were developed for early detection and differentiation of the four wheat-rust pathogen species, Puccinia graminis, P. striiformis, P. triticina, and P. recondita f.sp. secalis. To develop these assays, nuclear rDNA was used because it contains both a variable region (ITS1) to distinguish between species and a conserved region (28S) as an internal control for each assay. To demonstrate the assays were widely applicable, 16 samples of P. graminis from 4 countries and 11 U.S. states, 6 samples of P. recondita from 3 countries and 3 U.S. states, 7 samples of P. striiformis from 2 countries and 2 U.S. states, and 14 samples of P. triticina from 4 countries and 7 U.S. states were tested. To verify the assay did not show positive results for other rust fungi, rDNA from 10 different rust fungal species and wheat were tested. The results of this study show that in all cases the assays correctly identified only the rust species they were designed to identify. Also, the limits of detection were equivalent to 1-3 spores. Finally, six rusts unidentifiable in the field were sent to the laboratory and were correctly identified using the assay system, demonstrating a clear application of this work. We have also used the results from this work in experiments involving detection of P. graminis and Phakopsora pachyrhizi (Asian soybean rust) spores in rain. Further application of the results of this study could be used by scientists working on detection and identification of spores of rust fungi or by scientists developing disease-forecasting models.
Technical Abstract: Puccinia rust species are widespread pathogens of cereals and grasses that annually cause significant yield losses worldwide. Most spore types and early symptom morphology of these rusts are not useful in distinguishing between rust species. Therefore, duplex real-time PCR assays were developed for rapid detection of the four wheat-rust pathogen species, P. graminis, P. recondita f.sp. secalis, P. striiformis, and P. triticina, using TaqMan chemistry. Nuclear rDNA provided both a variable region (ITS1) to distinguish between species and a conserved region (28S) as an internal control. Specific ITS1 primer/probe sets exhibited high specificity and sensitivity with detection limits below 1 pg of rust DNA, equivalent to 1-3 urediniospores. The species-specific primer/probe sets exhibited high precision and reproducibility over a linear range of 5 orders of magnitude or greater. Specificity of the assays was tested across multiple isolates representing a range of races and forma specialis within a species. Additionally, assay specificity was evaluated by testing a range of other grass rusts as well as other fungi. The 28S primer/probe combination was successful in detecting all grass rust species tested within the duplex assays, validating the integrity of each assay. Rust species were identified within 30 min from the beginning of the assay, demonstrating one of the advantages of real-time PCR for detection of cereal rust pathogens. Finally, six visually unidentifiable rusts infecting several different grass species were correctly identified using the assay system and confirmed by DNA sequence analysis.