Researching Fusarium poae: its mycotoxines, chemotype and influence of oxidative stress triggers

Promotor: Geert Haesaert
Copromotors: Kris Audenaert, Sarah De Saeger, Monica Höfte
Collaborator: Adriaan Vanheule

Period: 08/2011-08/2015

Fusarium head blight (FHB) is a fungal disease that can occur on several cereals, including wheat. An important characteristic of FHB is that the disease is caused by a blend of up to 17 distinct species causing identical symptoms. The interest in FHB research is primarily fueled by the potential of FHB pathogens to produce mycotoxins. These mycotoxins are low-molecular organic compounds that can be toxic for humans and animals and as such implicate a serious health issue.
Thorough research at the Ghent University College recently attempted to shed light on the ever changing composition of the Fusarium complex. It was found that in Flanders, Fusarium poae has an important place within this disease complex. Up to recently, research in this field did not focus on F. poae, as other species were much more aggressive within the complex. Its rising presence, its nature as a secondary attacker and its potential to produce mycotoxins which could be much more toxic than those of other species, illustrate the large need for research on this topic.
The Fusarium poae research at the Ghent University College aspires to fill the gaps in the scientific community’s knowledge on this fungus. Field isolates that originate from several testing locations in Belgium are characterized pursuing a multidisciplinary approach of chromatographic, genetic and pathological methods. LC-MS/MS techniques are used to unravel the chemotype of the isolates. To this end, the Fusarium poae project has ties with the ‘Mytox’ Association Research Group (AOG), and research is carried out in the Bromatology lab at the Faculty of Farmaceutical Sciences at the University Ghent. The qualitative and quantitative production of toxins is examined under different conditions and influences of stress, such as fungicide treatment.
A further characterization of the field samples involves fungicide resistance assays of F. poae and the related species F. langsethiae to fungicides such as triazoles and strobilurines. Experiments to examine the physiological effects of fungicide treatments on both a macroscopic and a microscopic level are set up. This work is carried out in the broader framework of oxidative stress as a trigger for toxin production, which could be a central dogma in toxin production in all toxigenic fungi. To find a measure for aggressivity of the isolates, detached leaf assays are used. This technique also allows us to test several sources of resistance in wheat for its strength against F. poae. Again, microscopy visualization techniques are employed, this time to gain insight in the infection strategy of the fungus and the plant’s corresponding response. The last piece of the puzzle is filled in when AFLP-fingerprinting lets us unravel the genotype of the different isolates.
These different approaches of the Fusarium poae problem allow us to paint a fairly complete picture of the fungus, and as such meet the scientific community’s need for more knowledge on this organism with its importance increasing now more than ever.

SHORT TRAINING INITIATIVE

The Short Training Initiative (STI – VLIR-UOS) with as promoter Prof. dr. Ph. Sarah De Saeger is an intensive training on mycotoxin analysis. The main target group are researchers from developing countries working for the government, universities and NGOs or students from developing countries at the final year of their Masters degree program. This 9-day training is planned for the end of August, beginning September 2012.
Mycotoxin contaminations occur world-wide and represent one of the most challenging and prominent food safety threats. In terms of exposure and severity of chronic disease, especially cancer, mycotoxins appear at present to pose a higher risk than anthropogenic contaminants and pesticides. They seriously affect the agricultural economies of many countries, interfere or even prevent trade, reduce animal and animal product production and affect human health. Between 25% and 50% of all commodities, especially staple crops, are contaminated with mycotoxins. In developing countries, monitoring and enforcement (according to EU or US legislation) only occurs occasionally when commodities such as groundnuts and coffee are destined for export while it is mostly ignored for local goods. Agricultural commodities of inferior quality are kept for local use resulting in adverse and even fatal health effects. Fortunately, the awareness on the mycotoxin problem is growing in several developing countries.
This training session aims teaching the trainees most important analytical methodologies for mycotoxin analysis in food and feed. This will be done by practical exercises at the Laboratory of Food Analysis, Ghent University, Belgium.
Moreover, the awareness on the mycotoxin problem in developing countries will be further enhanced by providing the trainees with theoretical lessons given by different experts on the several aspects of the mycotoxin issue.
If you are interested, please send your curriculum vitae (name, date of birth, country of origin, institution, graduation degree, final year Masters degree, …) to sarah.desaeger@ugent.be.

MycoHunt: Rapid biosensor for the detection of mycotoxin in wheat

Promotor: Sarah De Saeger (coördinator UGENT), Mia Eeckhout (coordinator HOGENT)
Collaborator: Yirong Guo  (UGENT), Melanie Sanders (UGENT), Freya Martens (HOGENT)

Period: 01/09/2010-31/08/2013

MycoHunt is a project granted in the EU7 framework program for the development of a rapid online biosensor for the detection of mycotoxin in wheat.

The project aims to increase the competitiveness of a large group of “Small and Medium Enterprises (SME) Associations” in Europe by developing a cost-effective method to detect the contamination of deoxynivalenol (DON) in wheat grains. This mycotoxin forms a major threat in the food and feed sector of the European industry. A group of SME Associations, covering the relevant sectors and representing a vast number of sector SMEs, participate in the project. In this way, they will gain knowledge and resources to further exploit the results of the novel technology developed by providing a thorough sampling and measurement method of grain.

In the framework of the project the development of an online, non-destructive sampling apparatus for dust and other low molecular weight particles is targeted.  Another aspect of the research is the development of a specific sensor based on an immunoassay method using DON specific monoclonal antibodies. Beside the technological objectives, the research focuses on the determination of parameters (temperature, pressure, vacuum, etc.) affecting the sampling precision. Also different immobilizing methods for the DON specific monoclonal antibodies will be investigated. The determination of the cross-reactivity of these monoclonal antibodies against other trichothecenes, like 3-acetyl-DON, 15-acetyl-DON, nivalenol, is also an interesting point to consider.

The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under grant agreement n°243633.

Development and application of  molecularly imprinted polymers (MIP’s) in mycotoxin diagnostics

coordinators: Nicolas Gryson & Peter Maene
Copromoters: Sarah De Saeger & Peter Dubruel
Collaborators: Frederic Dumoulin

Period: 01/01/2009 – 31/12/2011
Grant: IWT-TETRA 080109

Fumonisin (FUM) is a mycotoxin that is mainly present in corn and derived products. The presence of fumonisin in food and feed products may pose a risk to human and animal health.

Current detection methods for fumonisins are based on the use of immuno-affinity columns. Due to several disadvantages related to the development (use of laboratory animals, long development stage) and the use of these columns (high cost, single use), there is an increased demand for alternative user-friendly detection methods.
The goal of this research project is to develop a quick test for the detection of fumonisin in corn products with the aid of molecularly imprinted polymers (MIPs).

A first step involves the development and characterisation of the specific MIP. Main variables in this process are the choice of the functional monomer molecule, the cross-linker molecule and the porogeneous solvent used during polymerisation. The characterisation basically implies the determination of the binding capacity.
In a second step, the procedure for fumonisin extraction from different corn products will be optimised, using a solid-phase extraction method. The optimised method should guarantee a recovery of 70 to 110 %. Based on those two steps, a quick test for fumonisin detection will be developed. This is a semi-quantitative test based on fluorescence detection.
The developed test will be an adequate tool for food and feed companies for the auto control of samples, in order to comply with the current Belgian and European legislation on food and feed safety. The test will be fast, simple, cheap and user-friendly. It will therefore be a convenient tool for all stakeholders in the corn supply chain in the prevention of fumonisin contamination.

Investigation of new analytical challenges for mycotoxin determination: Masked mycotoxins

Promoter: Carlos Van Peteghem
Copromoters: Sarah De Saeger, Dieter Deforce, Geert Haesaert, Mia Eeckhout
Collaborators: Marthe De Boevre, José Diana Di Mavungu, Peter Maene

Period: 01/08/2009 – 31/07/2013
Project number: BOF 05V00709, Research association project

Conjugated mycotoxins, in which the toxin is usually bound to a more polar substance such as glucose, are referred to as masked mycotoxins, as these substances escape established anlatytical techniques but can release their toxic precursors after hydrolysis. Little is known about the occurrence of these compounds in foodstuffs.

High-performance liquid chromatography (HPLC) combined with tandem mass spectrometry (MS/MS) offers a powerful tool for identification and characterization of mycotoxin conjugates. Derivatives can be identified due to their molecular mass and their collision induced dissociation (CID) fragmentation behavior, and unknown mycotoxin conjugates can also be identified with different MS techniques, namely quadrupole time-of-flight (Q-ToF) MS and Fourier transform (FT) MS.

The aim of this project is to evaluate the applicability of LC-MS/MS en high resolution MS tackle the problem of masked mycotoxins. Through this research project, an answer to the following questions will be formulated:

• Which mycotoxin conjugates are found in cereal-based food and feed?

• Is the occurrence of masked mycotoxins dependent on the cereal variety and genotype?

• When and how are conjugated mycotoxins formed?

• What is the share of masked mycotoxins to the total mycotoxin content?

• Do food processing technologies play a role in the occurrence of masked mycotoxins?

• Do mycotoxin conjugates contribute to overestimations of mycotoxin levels with commercial ELISA kits as compared to the traditional LC methods?

Assessment of the chemical risk associated with masked Fusarium toxin-containing

Coordinator: Sarah De Saeger
Copromoters: Carlos Van Peteghem, Mia Eeckhout, Geert Haesaert, Kris Audenaert
Collaborators: José Diana Di Mavungu, Marthe De Boevre, Peter Maene

Period: 01/03/2010 – 31/02/2012
Project number: RT 09/3 MYCOMASK, FOD Volksgezondheit, Veiligheid van de Voedselketen en Leefmilieu

Conjugated or masked mycotoxins first came to attention in the mid-1980s, when animals fed with apparently low mycotoxin contaminated feed showed high severity of mycotoxicosis. The unexpected high toxicity was attributed to the presence of conjugated forms of mycotoxins, possibly generated by the plant metabolism, which were undetected by commonly used analytical methods. These derivatives could be hydrolysed to the precursor toxins in the digestive tracts of the animals or could exert toxic effects comparable to those imputable to free mycotoxins. Little is known about the occurrence, bioavailability and metabolism of these compounds so that a comprehensive study is needed.

This project aims at the following objectives:

1. Develop reliable extraction, purification and quantification methods based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of free and masked Fusarium toxins in cereals and cereal-based foods and feeds;

2. Provide quantitative data concerning the occurrence of masked Fusarium toxins and the circumstances in which they are formed, mainly the glycosylated metabolites of deoxynivalenol (DON), zearalenone (ZEN) and T-2 toxin (T-2) in cereals and cereal-based foods and feeds;

3. Assess the extent in which animals and humans are exposed to masked mycotoxins and carry out a preliminary risk evaluation.