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Sorting through Fusarium complexities

A study to identify FHB species generates some intriguing results and an improved diagnostic toolkit.

April 22, 2024  By Carolyn King


Mohamed Hafez and a summer student with some of the many Fusarium isolates analyzed during this project. Photo courtesy of Reem Aboukhaddour, AAFC-Lethbridge.

Sometimes going the extra mile can take you much farther than expected. That was Reem Aboukhaddour’s experience in her recent study of Fusarium head blight (FHB) species in wheat.

Her study was part of a major, five-year project led by plant pathologist Kelly Turkington with Agriculture and Agri-Food Canada (AAFC). The project investigated crop management options to lessen the impact of FHB in wheat. It looked into the effects of crop rotation, residue management, row spacing, seeding rate and fungicide timing on FHB, leaf disease and yield of spring wheat. It took place at nine sites in five provinces and started in 2018. 

Turkington asked Aboukhaddour, who leads the cereal pathology lab at AAFC-Lethbridge, and Adam Foster, a plant pathologist with AAFC-Charlottetown, to detect and quantify F. graminearum in grain and node samples from the different treatments. Aboukhaddour’s research team analyzed the samples from the project’s four western Prairie sites – Lethbridge, Lacombe and Beaverlodge, Alta., and Scott, Sask. – and Foster’s group worked on samples from the five sites in the eastern Prairies and Eastern Canada.

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At first, Aboukhaddour was a bit worried by the amount of work that would be involved, especially since she had never worked on Fusarium before. But she very quickly realized that this task was a generous opportunity: it provided her team with a treasure trove of wheat samples for pathogen analysis.

“My team and I brainstormed about how to plan the work and whether we could also expand the use of the samples beyond detecting F. graminearum,” she says. “We decided to look at all the fungi in the samples and look at Fusarium in depth. We thought that having five years of all this information might guide us into some new or interesting research areas.”

All fungal species
Over the five years of the project, Aboukhaddour’s team found a total of 27 species belonging to 15 genera, based on studying the pathogen cultures under a microscope and using molecular testing.

“The most frequently isolated genera from the grain and nodes were Alternaria, Parastagonospora and Fusarium. Those three genera represented over 90 per cent of all isolates. Alternaria species are not known to be major plant disease concerns, although some species can cause minor disease in cereals,” she notes.

“However, the Parastagonospora species we detected were mainly Parastagonospora nodorum and its sister species Parastagonospora pseudonodorum, which cause septoria nodorum. This is an important leaf spot disease, but people sometimes forget that these pathogens can also cause glume blotch. As someone who works on leaf spots, finding Parastagonospora nodorum on both grain and node samples was really interesting.”

Out of the study’s Parastagonospora findings came a new project led by Aboukhaddour to investigate Parastagonospora nodorum and related species in wheat.

FHB disease complex

“Fusarium head blight is a disease complex caused by many different species. F. graminearum is the predominant FHB-causing pathogen in Canada and worldwide. But many other Fusarium species can be involved, and those species are varied in their response to fungicides, weather conditions and many other factors. Besides causing significant yield losses, FHB pathogens reduce grain quality and may produce harmful mycotoxins,” explains Aboukhaddour.

“It’s important to know which Fusarium species are involved in causing FHB in a crop because different species may be affected differently by management measures. Also, different species produce different mycotoxins. These mycotoxins can affect the pathogen’s virulence on the plant, and some of these mycotoxins may affect food and feed safety. For example, DON (deoxynivalenol) is a key mycotoxin of concern because it can cause toxicity to animals and humans. F. graminearum produces DON and so does F. culmorum.” 

Aboukhaddour’s team cultured isolates from the grain and node samples and examined the cultures under a microscope to identify all the Fusarium species. Then, they confirmed the species identification through molecular tests.

A better diagnostic tool
Some Fusarium species are extremely difficult to tell apart based on their morphology in lab cultures and under the microscope, but a further challenge in identifying Fusarium species is the changing scientific opinions about the species classifications. 

Fusarium taxonomy has been a controversial issue and many taxonomical concepts have been used to define species limits for Fusarium. In recent decades, the application of the phylogenetic species concept based on DNA sequencing has resulted in many taxonomic revisions within the genus Fusarium,” Aboukhaddour says. 

“For example, in the early 2000s, experts determined that what had been identified as F. graminearum was actually comprised of 16 different species, collectively known as the F. graminearum species complex. One of the 16 has been designated as F. graminearum itself.”

Fortunately, at the start of this study, Aboukhaddour added Mohamed Hafez to her team. Hafez had previously studied Fusarium species in cereals and pulses and had experience in developing molecular diagnostic kits for differentiating certain Fusarium species.

Hafez pointed out that the markers developed before 2000 were not very good at accurately differentiating between the 16 FHB species. As a result, Aboukhaddour’s team ordered various FHB species from culture collections, tested the available markers and confirmed the markers were not able to accurately tell all the species apart.

So, through a lot of testing and tweaking, the team developed a set of quantitative PCR markers for the four most abundant wheat-associated Fusarium species in Canada: F. avenaceum, F. culmorum, F. graminearum and F. poae. Quantitative PCR (qPCR) tests not only determine whether or not a particular pathogen is present in a sample but also how much of that pathogen is present. 

The team also optimized the protocol for using this set of qPCR assays, and they validated that the assays reliably and accurately differentiated the four species from each other and from other Fusarium species. 

Developing this diagnostic toolkit took about a year. Aboukhaddour had hoped to also develop markers for two more Fusarium species, but that additional work was put on hold due to pandemic restrictions on lab work.

Aboukhaddour’s team immediately shared the toolkit with Foster and other colleagues working on Fusarium, and they published the toolkit in 2022 so researchers anywhere can now use it.

“This diagnostic tool has a really powerful potential for improving our understanding of the FHB complex,” says Aboukhaddour. “For instance, by collecting accurate information over time about how much of a particular pathogen is present in your samples, you can track changes in the pathogen populations and see which is the main pathogen causing FHB. You can look for patterns to see how changes in the proportion of the different species relate to differences in things like weather conditions, cultivars or management practices.”

For all five years of the project, Aboukhaddour’s team used the qPCR tests for F. graminearum for Turkington to use in his analysis.

However, for the three other species, the team was only able to run the qPCR tests for the samples collected in 2018, again because of pandemic restrictions. With data for only one year, they can’t draw any firm conclusions about the effects of the different management practices on the quantity of F. avenaceum, F. culmorum and F. poae.

Key Fusarium species findings
Aboukhaddour summarizes the Fusarium species results for the four western Prairie sites. 

“We found three species on the grain and nine on the nodes. F. avenaceum was the most abundant species in node samples, whereas F. poae was the most abundant in grain samples.

F. avenaceum was recovered from node and grain samples in all locations. F. culmorum was exclusively recovered from node samples in all four locations and was the most abundant species in Beaverlodge.

F. graminearum was recovered at low frequency from node and grain samples collected from Lethbridge and Scott and was not detected in Beaverlodge or Lacombe.”

Aboukhaddour notes that F. graminearum tends to be much more predominant in regions with moister growing conditions, such as Manitoba, compared to Alberta. “Maybe in Alberta, we should also look at F. avenaceum, F. culmorum and F. poae – along with F. graminearum – because those three species seem to also be an important part of the FHB complex here.” 

Learning more about the impacts of these other species on wheat yields and mycotoxin levels might be especially important if the coming years bring more of the hotter, drier conditions that seem to favour species like F. poae.

Aboukhaddour thinks it might also be worth looking into crop management effects on these other FHB species. For instance, she notes that recent research led by Xiben Wang at AAFC-Morden indicates that different FHB species on barley might have different sensitivities to fungicides.

Going above and beyond
“This whole study was really born by accident. I’m happy I went beyond the original task of detecting F. graminearum. The samples from Kelly’s project were hugely valuable for us, for this study and beyond. Now we have this diagnostic tool for Fusarium that can help a lot of other people. And my team has a big project on Parastagonospora nodorum about an aspect that I hadn’t considered very much before,” Aboukhaddour says.

“By going beyond the original task, we opened a door to new questions and new research areas that could help toward a better understanding of important cereal diseases in the western Prairies.”

Funding for this FHB study came via Turkington’s project, which was supported by the Wheat Cluster (Canadian Wheat Research Coalition and AAFC). Aboukhaddour’s lab was also the recipient of additional funding from AAFC, Alberta Grains (formerly Alberta Wheat) and Saskatchewan Wheat Development Commission. 

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