Development and evaluation of real-time PCR techniques for the early detection of three airborne fungal pathogens of wheat
Creators: Thirugnanasambandam, Ilakkiya
Publisher: name: University of Alberta Library
Year: 2023

<p>Wheat is a staple food crop with 760 million tonnes consumed globally in 2020 with Canada being a major producer. Airborne fungal pathogens pose a severe threat to wheat growers all over Canada. New isolates of pathogens evolve through mutations which may become resistant to control measures and can cause significant yield losses. To minimize these losses, early detection of these pathogens is needed. This research aims to develop highly specific and sensitive real-time immuno-PCR (RT-iPCR) assays for the detection of three different pathogenic fungi infecting wheat: Pyrenophora tritici-repentis (Ptr), Fusarium graminearum (Fg), and Puccinia striiformis f.sp.tritici (Pst) causing tan spot, Fusarium head blight, and stripe rust of wheat, respectively. RT-iPCR resulted in a limit of detection of 1, 188, and 938 spores for Ptr, Pst, and Fg, respectively without requiring DNA extraction as this method measures spores directly. RT-iPCR sensitivity was improved 5-, 12-, and 30-fold for Fg, Pst, and Ptr, respectively compared to its corresponding enzyme-linked immunosorbent assays (ELISA). However, specificity remained a challenge for the RT-iPCR assay when assessed through cross-reactivity tests. All three antibodies evaluated reacted strongly with non-target antigens suggesting high cross-reactivity. An alternative and more established quantitative PCR (qPCR) technique was investigated to further evaluate the accuracy of the more novel RT-iPCR technique developed here. Additionally, an approach was developed to determine spore DNA extraction efficiency for qPCR which represents a methodological gap in most qPCR spore measurements. Using fungal reproductive biology a formula was derived to calculate spore numbers from the DNA quantified by the qPCR. The calculated spore numbers were compared to pure spore cultures counted under a microscope to determine DNA extraction efficiency. Ultimately, this approach will help plant pathologists quantify pathogens more accurately. DNA extraction efficiency determined for Fg, Pst and Ptr spores are 5 ± 0.1%, 14 ± 0.7% and 290 ± 36%, respectively. qPCR resulted in a limit of detection of 3, 400, and 800 spores for Ptr, Pst, and Fg respectively after being corrected to DNA extraction efficiency. Finally, air samples were collected over the 2022 field season from two research fields located at the Agriculture and Agri-Food Canada – Lethbridge Research and Development Centre. The two fields were inoculated with Pst and samples were evaluated and compared using three detection techniques; RT-iPCR, qPCR and microscopy. Overall, the three techniques showed similar trends in Pst spore numbers over time. Microscopy and RT-iPCR showed higher measurement variability compared to qPCR, likely due to the challenges associated with spore identification at the species level using microscopy and known cross-reactivity associated with RT-iPCR. The established technique of qPCR appears to be the most accurate technique for fungal spore detection if extraction efficiencies are considered and used to correct reported spore numbers. While more research is needed to refine the RT-iPCR technique to improve specificity, this research is an important step forward and will help in efforts to prevent the spread of plant diseases through the early detection of fungal spores.</p>