Metabolome of two major fungal contaminants of wheat: identification of new toxic metabolites – Newmyco

Since the launch of the project, our work has focused on: - producing labelled grains and determining the conditions for carrying out such double isotopiclabelling during F. graminearum infestation - characterizing the metabolome of Penicillium verrucosum during its development on grains - Evaluate the depth of analysis necessary for the characterization of the fungal transcriptome in planta

A number of preliminary trials have thus been carried out in close partnership between INRA Clermont and the CEA to define the optimal conditions for cultivation in confined enclosure. Indeed, confined culture for the double isotopic labeling of Fusarium graminearum infested plants proved to be more complex than expected. Indeed, the fungal infection causes a considerable reduction in the weight of the grains and, considering the space constraints in the enclosures and gas consumption, greatly limits the weight of the biological material available at the end of the test. several tests had to be carried out to test different varieties of wheat and to maximize the use of the labelling chambers. A first labelling of 50% 13C and 100% N15 was successfully performed. Since the beginning of the project, the work carried out has been able to characterize the metabolome of P. verrucosum. The extracts of the cultures made on labeled grains were analyzed by high resolution mass spectrometry. By isotopic labeling, secondary metabolites were specifically detected and their crude formulas were characterized. Then, the characterization of new secondary metabolites was assisted by the generation of molecular networks of MS / MS similarities. The study of P. verrucosum detected 98 secondary metabolites. Of these, 80% are unknown according to current databases. The generation of molecular networks from the secondary metabolome made it possible to demonstrate a group of 25 compounds behaving similarly in mass spectrometry. Sixteen of these compounds have been identified as derivatives of fungisporins, metabolites suspected of interfering with the aerial growth of fungi. This work has been the subject of a publication and several presentations at international and national congresses.

In the coming months, the F. graminearium metabolome will be characterized, using a labeled kernel culture (as was done with P. verrucosum) but also using labeled grains during fungal infestation to analyze the metabolome in planta. At the same time, trials to study the varietal impact on the fungal metabolome will begin, with a number of field samples already collected by the project's industrial partner.

1 publication published (Hautbergue et al., Evidencing 98 secondary metabolites of Penicillium verrucosum using substrate isotopic labeling and high resolution mass spectrometry. Chromatogr B analyt Technol Biomed Life Sci, 2017, doi.org/10.1016/j.jchromb.2017.03.011) 1 publication submitted 4 communication to scientific congresses 2 articles of scientific vulgarization 1 presentation in the framework of «my thesis in 180 seconds«, Miss Hautbergue having been a national finalist.

Fungi are frequent contaminants of cereals worldwide, especially in wheat, the most important cereal for human food in Europe. According to environmental conditions and their own physiological specificities, fungi can develop on living plants, in the field or during peri-harvest period as well as during storage in case of drying failure or re-moistening.
Such a development can lead to plant diseases and loss in production yields; it can modify organoleptic and nutritional qualities of harvested grains; alter their technological properties and finally, also lead to the accumulation of toxic compounds named mycotoxins. Secondary fungal metabolism is very active but unfortunately weakly known since it is admitted that, in best known fungal species such as Fusarium graminearum, only 20% of secondary metabolites are characterized.
Within this context, the main objective of this project is to identify new toxic metabolites that can be produced by 2 major fungal species: Fusarium graminearum, which contaminates wheat in the fields, and Penicillium verrucosum, which is the most important contaminant during storage of grains. For that, the complete metabolome of these two species will be characterized using a new analytical strategy based on the comparison by high-resolution mass spectrometry of secondary metabolites profiles obtained from fungi grown on differently isotopically enriched wheat grains. The new metabolites will be purified and further characterized for their structure and toxicity.
Worldwide surveys demonstrated that known mycotoxins are very widespread in cereals, found in about 70% of analysed samples., but with levels of contamination that can strongly vary, according to climatic conditions and agricultural practices. At the European level, only few of them are subjected to regulation in human foods and recommendations in animal feeds.
It is obvious that, beyond well-characterized mycotoxins, still unknown fungal toxic compounds may contaminate wheat.
Moreover, the improvements of analytical methods developed for mycotoxin analysis raised some important questions regarding these contaminants:
- Part of the toxins is conjugated to plant components and is therefore not directly detected.
- Other compounds are produced simultaneously with regulated mycotoxins. Among these molecules, some display a toxicity that can equal or even exceed the one of regulated toxins.
These data highlight that the fungal secondary metabolism, leading to mycotoxin contamination of cereals is still poorly known.
The specific objectives of the project will be:
- To characterize the secondary metabolome of Fusarium graminearum during infection of wheat living plants and grains
- To characterize the secondary metabolome of Penicillium verrucosum during grain contamination.
- To compare the impact of the varietal resistance of wheat (sensitive, intermediate and resistant) on this secondary metabolome.
- To identify new mycotoxins among major secondary metabolites that will be identified.

To reach these objectives, the project will benefit from the expertise of
- two INRA research teams specialized on fungal metabolism (primary and secondary metabolism) and fungal toxinogenesis (Team biosynthesis and toxicity of mycotoxins, UMR INRA-INPT Toxalim) as well as on plant response to fungal infection (team cereal diseases, UMR INRA 1095, Clermont Ferrand)
- an industrial partner (Syngenta France) specialized in selecting of new wheat varieties
- an analytical platform dedicated to trace contaminants analysis (MetaToul-AXIOM platform, UMR1331 INRA-INPT Toxalim).
- the CEA Cadarache that will be in charge of the production of stable isotope plants and grain labeling and will act as a service provider for the project.