Ilakkiya Thirugnanasambandam | ALES Graduate Seminar

Event details: A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES MSc Final Exam Seminar by Ilakkiya Thirugnanasambandam. This seminar is open to the general public to attend.

Zoom Link: https://ualberta-ca.zoom.us/j/91429075780?pwd=cXRzeWp4S05XcWZnSktjUm1Xd3B2dz09

MSc with Drs. Nat Kav and Jonathan Challis.

Thesis Topic: Development and comparisons of real-time PCR techniques for the early detection of three airborne fungal pathogens of wheat

Abstract:

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 taken and can cause significant yield losses. To minimize these losses, early detection of these pathogens is needed. This research aims to develop a highly specific and sensitive real-time immuno-PCR (RT-iPCR) assay 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 involving complex procedures of DNA extraction as this method directly measures the spores. The sensitivity was improved 5×, 12×, and 30× for Fg, Pst, and Ptr respectively with RT-iPCR over its corresponding enzyme-linked immunosorbent assays (ELISA); However, specificity remained a challenge when assessed through cross-reactivity assays. All three antibodies evaluated had a strong reaction with non-target antigens suggesting high cross-reactivity. An alternative and more established quantitative PCR (qPCR) was investigated and an existing research gap was studied. A potential approach was developed to determine DNA extraction efficiency using fungal reproductive biology and a formula was derived to calculate spore numbers from the quantified DNA amount in the qPCR. This calculation would help plant pathologists quantify pathogens more accurately. DNA extraction efficiency determined for Fg, Pst and Ptr spores are 5%, 14% and 290% 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, samples from two fields from Agriculture and Agri-Food Canada – Lethbridge Research and Development Centre were evaluated and compared using three detection techniques such as RT-iPCR, qPCR and microscopy. These techniques would help in early detection of fungal spores and prevents the spread of plant diseases.