A quantitative PCR assay for detection and quantification of Fusarium sambucinum

Authors: THOMAS, WILLIAM - Oregon State University; BORLAND, THEODORA - Oregon State University; BERGL, DARBY - Oregon State University; CLAASSEN, BRIANA - Oregon State University; FLODQUIST, TIMOTHY - Oregon State University; MONTGOMERY, ALEXANDRIA - Oregon State University; Rivedal, Hannah; WOODHALL, JAMES - University Of Idaho; OCAMB, CYNTHIA - Oregon State University; Gent, David - Dave

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/22/2022
Publication Date: 4/29/2022
Citation: Thomas, W.J., Borland, T.G., Bergl, D.D., Claassen, B.J., Flodquist, T.A., Montgomery, A.S., Rivedal, H.M., Woodhall, J., Ocamb, C.M., Gent, D.H. 2022. A quantitative PCR assay for detection and quantification of Fusarium sambucinum. Plant Disease. 106:2601-2606. https://doi.org/10.1094/PDIS-02-22-0269-RE.
DOI: https://doi.org/10.1094/PDIS-02-22-0269-RE

Interpretive Summary: The fungus Fusarium sambucinum causes disease on multiple plants, including hop, potato, alfalfa, and numerous cereal crops. Current diagnostic methods for the fungus are costly, time consuming, and require specialized training to be able to identify the pathogen. We sought to develop a DNA-based assay that could quickly and reliably detect low levels of the fungus in infected plant tissue and other samples. We identified a genetic region in the fungus that appeared unique to this organism and used this region to develop an assay that enables both detection and quantification of the fungus in environmental samples. Our assay was more sensitive than current approaches, did not give false positive results with closely related organisms, and was robust when tested against strains of the fungus from different regions of the world and diverse hosts. This assay will be useful in diagnostic laboratories and research contexts, accelerating routine disease diagnoses and research discoveries.

Technical Abstract: Fusarium sambucinum is an ascomycete that has been isolated from a broad range of plant hosts including hop (Humulus lupulus L.), where it acts as a causal agent of Fusarium canker, a disease that can impact cone quality and yield in severe cases. Current diagnostic methods rely on isolation of the fungus from plant tissue, a time- and resource-intensive process with limited sensitivity. Our objective was to develop a rapid and sensitive diagnostic tool to detect and quantify F. sambucinum in plant tissues. Using a modified random amplified polymorphic DNA PCR assay, we identified a F. sambucinum-specific marker that serves as the target in a TaqMan (hydrolysis) probe qPCR assay that can be used to detect F. sambucinum DNA in a background of plant genomic DNA. When used to screen 52 isolates of F. sambucinum and isolates representing 13 other Fusarium species, the assay was robust in detecting F. sambucinum while discriminating between F. sambucinum and closely-related Fusarium species. Furthermore, this assay reliably detects as little as 1 pg of F. sambucinum DNA in a background of total genomic DNA from plant tissue. Within-sample comparisons of this qPCR assay with traditional cultural isolation methods demonstrated the greater sensitivity of the qPCR-based method for detection of F. sambucinum. When used to screen 220 asymptomatic stem samples, the qPCR assay detected F. sambucinum in 100 samples (45.5%); by comparison, F. sambucinum was detected in only 3 samples (1.3%) by culturing methods. Moreover, quantification of F. sambucinum DNA was possible for 60 of these samples, indicating the utility of the qPCR assay for early detection. This assay should be useful in diagnostic and epidemiological applications to detect and quantify F. sambucinum from multiple hosts and environmental samples.