Stilbenoids as promising environment-friendly natural products to minimize the contamination of wheat and grape with toxigenic fungi and associated mycotoxins – StilDeTox

The StilDeTox project was driven by the necessity to develop an effective and sustainable solution to manage the risk of mycotoxins in the cereal and wine sectors and to ensure the safety of cereal products and grape derivatives. The promising potential of grapevine co-products as sources of bioactive compounds, and more specifically bioactive stilbenoid extracts, was investigated to identify effective natural antifungal and anti-mycotoxin solutions. To achieve its goal, StilDeTox implemented a strategy combining fundamental and applied studies. The StilDeTox project was organized into three chronologically sequential tasks that incorporate an integrated approach, from the production and selection of stilbene extracts (Task 1) to the identification of active molecules and elucidation of their mechanisms of action (Task 2), and finally to the in planta efficacy evaluation on wheat and ex vivo on wheat leaves and grape berries of the active extracts and optimization of the production process (Task 3).

The StilDeTox project's results can be categorized into three main areas.
(1) The project provided original and central knowledge on the phytochemistry of stilbenoids from grapevine co-products (different parts of grapevines from various genetic origins) and, on the ability of this class of phenolic compounds to counteract the fungal growth and production of mycotoxin by two pathogens of major concern in the European agriculture sector. F. graminearum seems to be much more sensitive to the studied extracts compared to A. carbonarius. Omics approaches (transcriptomics and metabolomics) were used to investigate the modes of action of molecules potentially involved in the bioactivity of these extracts, particularly stilbene tetramers, showing that they affect the fungal pathogen's primary and secondary metabolisms.
(2) StilDeTox delivered stilbenoid extracts with a confirmed ability to effectively and significantly decrease the development of F. graminearum and its DON production in wheat. For A. carbonarius, the antifungal and anti-mycotoxin effects of the extracts were less drastic, necessitating the development of a specific formulation to ensure good bioavailability of the products.
(3) The project delivered pre-commercial environment-friendly plant care product ready for formulation, with demonstrated in planta efficacy targeting F. graminearum and its DON production to be used in the cereal sector.
In conclusion, grapevine co-products, especially roots, are a rich source of oligomerized stilbenes with promising antifungal and anti-mycotoxin properties. This paves the way for their valorization in an eco-responsible and integrated approach to combat phytopathogenic fungi and associated mycotoxins.

Biocontrol strategies are key initiatives that the cereal and wine industries are investing in as part of the French Ministry of Agriculture's EcoPhyto 2030 strategic plan, aiming for increased and improved production. This work will contribute to developing alternative solutions for these two crucial sectors in France.
Currently, the project is at TRL 3-4, moving from the basic principle to proof of concept for application. Studies focused on understanding the mechanisms of action of stilbenoids will, in collaboration with private partners, enable the development of specific and tailored formulations.
Since fungal species don't exist in isolation on wheat ears or grape berries but are constantly competing with other microbial species (including those in the Fusarium complex for Fusarium Head Blight), short-term studies are planned to assess the efficacy of these extracts on multiple fungal species, both alone and in combination, in addition to formulation efforts.
In the longer term, beyond the in planta work on the antifungal and anti-mycotoxin activities of the developed formulations, studies on indirect efficacy will be necessary. This includes stimulating the plant's natural defenses and examining the surrounding plant and soil microbiota (bacterial and fungal communities).
This research will advance the project's maturity level, leading to the development of sustainable biocontrol solutions by valorizing co-products from the wine sector.

Ahmed O. S. , Tardif C., Rouger C., Atanasova, V., Richard-Forget F., & Waffo-Téguo P. (2022). Naturally occurring phenolic compounds as promising antimycotoxin agents: Where are we now? Compr Rev Food SciFood Saf,1–37, doi.org/10.1111/1541-4337.12891

Trang Minh Trah, Vessela Atanasova-Penichon, Charles Tardif, F. Richard-Forget. Stilbenoids as Promising Natural Product-Based Solutions in a Race against Mycotoxigenic Fungi: A Comprehensive Review. Journal of Agricultural and Food Chemistry, 2023, 71 (13), pp.5075-5092. ?10.1021/acs.jafc.3c00407?. ?hal-04095931?

Omar S Ahmed, Faiza Tahanout, Gilberte Marti-Mestres, Jean-Frédéric Weber, Pierre Waffo-Téguo. An automated accelerated salting-out assisted solvent extraction (A-ASASE) of stilbenoids from Vitis vinifera L. branches: False proof or a proof of concept?. Talanta, 2024, 266, pp.124935. ?10.1016/j.talanta.2023.124935?. ?hal-04550128?

Tardif, Charles; Rouger, Caroline; Miranda, Julie; Ahmed, Omar; Richard-Forget, Florence; Atanasova, Vessela; Waffo-Teguo, Pierre (2024) Targeting of antifungal metabolites from grapevine by-products by UPLC-HRMS/MS approaches using bioactivity-based molecular networking. J Agric Food Chem. 72 (17) 9621-9636. doi: 10.1021/acs.jafc.3c09531.

Phytopathogenic fungi are among the dominant causal agents of crop diseases. In addition to direct losses related to yield reduction, some fungi produce mycotoxins harmful to humans and animals. In Europe, cereals and grapevine are the most concerned crops. The deoxynivalenol mycotoxin producer, Fusarium graminearum, and the ochratoxin A producer, Aspergillus carbonarius, are the most frequently found in cereal and grape crops, respectively.
Although good cultural practices have been proposed to minimize the contamination of wheat with deoxynivalenol and of grape berries with ochratoxin, they are not sufficient to guarantee the compliance of harvests with the EU regulations. Combined with tillage, crop rotation and cultivar choice for wheat, the use of fungicide is a key factor in the integrated management strategies aiming at controlling mycotoxins. However, according to the growing commitment of European and National governments for limiting chemical inputs, biocontrol is a promising solution to be used instead of or in complement to chemical treatments. The French National Ecophyto plan claims to gradually reduce chemical treatments, hence stimulating the development and use of all novel practices including biocontrol. The future of agriculture will require the development of new environmental-friendly agronomical strategies guaranteeing the safety of food and feed. Among these strategies, the development of products issued from natural sources to protect crops against fungal pathogens and their mycotoxin production will contribute to decrease the use of chemical fungicides. In this context, the goal of StilDeTox is to characterize natural stilbenoid extracts (STEs) from vine by-products to be used in integrative fungal/mycotoxin control strategies. The project is organized in three tasks and will implement an integrated approach, from the production and screening of the STEs (task 1), the elucidation of the mechanisms of action (task 2) to the assessment of the in planta efficiency of formulated solutions and optimization of the production process (task 3). Environmental preoccupations will drive the project: identification of alternatives to chemical fungicides, zero-solvent extraction procedures, valorization of grapevine by-products.
To achieve these objectives, StilDeTox associate three academic and one private partners selected for the diverse and complementary expertise and skills that they collectively bring into the project. The three academic partners are INRA, UR 1264 MycSA, which research programs focus on the understanding of the regulation of toxin biosynthesis by fungi, UR Oenology EA 4577, USC 1366 INRA, MIB which research programs target the isolation, characterization and evaluation of biological activities of phenolic compounds from grapevine/wine and, INRA, UMR 1065 SAVE specialized in developing integrated and sustainable protection strategies of the vine. The private partner is BioLaffort company which has expertise in plant product extraction and vegetal protection.
The first innovation of StilDeTox lies in its guiding challenge using vine-production waste to address significant public health and economic issues. StilDeTox will develop innovative methodologies starting from the recycling of vine industry by-products using ecological technologies and ending with the proposition of biofungicide formulations. A second major innovation lies in the multidisciplinary and transdisciplinary approach that will be implemented. Basic studies and applied approaches will be combined, allowed by the complementarity of the expertise gathered in the project’s consortium.