Performance Story: Establishment of LC-MS/MS based Mycotoxin/Deoxynivalenol (DON) Diagnostic platform for FHB research and breeding programs
Fusarium head blight (FHB), caused by Fusarium spp., is a destructive disease of small grain cereals, such as wheat, barley, oat and canaryseed. Apart from grain yield losses and reduced baking and seed quality, a major concern with FHB is crop contamination with Fusarium-produced trichothecene mycotoxins, specifically deoxynivalenol (DON), also known as vomitoxin. These mycotoxins accumulate in the grain making it unfit for consumption by humans and animals. Significant DON contamination may render a crop unmarketable, or reduce the market value by 40-65%.
Breeding productive cultivars with high disease resistance and low mycotoxin contamination is a priority for wheat breeders. However, measurement of DON content is not always included in breeding programs due to the lack of efficient quantification methods. Some methods are easy-to-use, but they lack the needed accuracy and sensitivity, such as enzyme-linked immunosorbent assay (ELISA); while some chromatographic-based methods have relatively high accuracy and sensitivity compared to ELISA, but require complex extraction and cleanup steps and longer running time, which are not cost-efficient and environmentally friendly.
In this study, a tandem mass spectrometry (MS/MS) method was established, which employed a one-step acetonitrile extraction protocol and flow injection analysis (FIA)-MS/MS method (i.e. no analytical column) to reduce the complexity, cost and time. This method is designed for FHB breeding programs or DON quantification for other purposes that require a fast, high-throughput DON phenotyping, but provides relatively high selectivity, accuracy and sensitivity compared to existing assays. This method has been fully validated according to the US Food and Drug Administration (FDA) Guidance for Bioanalytical Method Validation, including selectivity, linearity, accuracy, precision, recovery, matrix effects, stability and dilution integrity. With the ease of use, high sensitivity and accuracy, this high throughput DON quantification method will increase breeding efficiency and accelerate screening progress in the selection of FHB resistant germplasm.
In addition, researchers have developed a state-of-the-art analytical method to simultaneously quantify DON and its derivatives: 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and DON-3-glucoside (D-3-G); and the toxins: nivalenol (NIV), HT-2 and T-2. This multi-mycotoxin quantification method will help scientists to address some emerging issues related to Fusarium mycotoxins, such as monitoring aggressive chemotype changes, detecting new types of mycotoxins and addressing the masked/modified mycotoxin issue in Canadian varieties.
With these developments and findings, the team has started to provide mycotoxin testing for their own lab and collaborators to support FHB relevant projects, including FHB breeding, fungicide timing determination, disease surveys, mycotoxin degradation by insects, and postharvest grain and food contamination. They will continue to seek potential collaborators and expand our mycotoxin diagnostic library.