# Integrated metabolomic and transcriptomic analysis to elucidate the antifungal and antimycotoxin mechanisms of natural stilbenoids against Fusarium graminearum

Trang Tran-Minh, Nadia Ponts, Stéphane Bernillon, Pierre Waffo-Téguo, Caroline Rouger, Florence Richard-Forget, Vessela Atanasova

<figure><img src="image_1.png" /></figure>

## To cite this version:

Trang Tran-Minh, Nadia Ponts, Stéphane Bernillon, Pierre Waffo-Téguo, Caroline Rouger, et al. Integrated metabolomic and transcriptomic analysis to elucidate the antifungal and antimycotoxin mechanisms of natural stilbenoids against Fusarium graminearum. Microbiological Research, 2026, 303, pp.128370. 10.1016/j.micres.2025.128370. hal-05486454

HAL Id: hal-05486454

https://hal.inrae.fr/hal-05486454v1

Submitted on 11 Feb 2026

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

Check for
updates

# Integrated metabolomic and transcriptomic analysis to elucidate the antifungal and antimycotoxin mechanisms of natural stilbenoids against *Fusarium graminearum*

Trang Tran-Minh<sup>a,1,2</sup>, Nadia Ponts<sup>a</sup>, Stéphane Bernillon<sup>a</sup>, Pierre Waffo-Téguo<sup>b,c</sup>,
Caroline Rouger<sup>b,c,d</sup>, Florence Richard-Forget<sup>a</sup>, Vessela Atanasova<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Antimycotoxin

Biofungicide

*Fusarium graminearum*

Stilbenoids

Trichothecenes

Viticulture waste

## ABSTRACT

Climate change is driving a rise in the contamination of crops by mycotoxins, threatening global food security. If agrochemical use is to be reduced, environmentally friendly antimycotoxin solutions are urgently needed. This study aims to elucidate the antifungal and antimycotoxin modes of action of stilbenoid molecules from vine by-product extracts against *Fusarium graminearum*, the primary causal agent of *Fusarium* head blight in wheat and a main producer of type B trichothecene (TCTB) mycotoxins. *E*-resveratrol (RES), a monomer, and *E*-vitisin B (VIT), a tetramer, were selected as the targeted stilbenoids due to their predominance in vine extracts. VIT (8 µM) showed greater efficacy than RES (35 µM) in inhibiting spore germination, biomass production, and TCTB yield. To better understand their modes of action, a multi-omics approach combining mRNA-seq-based transcriptomics and LC-MS/MS-based untargeted metabolomics was employed. mRNA-seq data evidenced that VIT exposure altered the expression of nearly half of *F. graminearum* genes, whereas RES induced only minimal transcriptomic changes. Integrated with metabolomics data, our findings suggest that VIT's modes of action involves: (1) disruption of the fungal cell wall and plasma membrane via downregulation of sphingolipid metabolism, (2) inhibition of sporulation and hyphal growth, and (3) both direct and indirect interference with TCTB biosynthesis. These findings support the potential of viniculture waste-derived compounds as biofungicides, offering a dual benefit of reducing cereal mycotoxin contamination and valorising underused vine biomass.

## 1. Introduction

Wheat (*Triticum aestivum* L.) is the main staple cereal in Europe, accounting for 46.3 % of all cereals harvested in 2023 (EUROSTAT, 2024) and providing approximatively 20 % of the daily protein and calorie intake for human consumption (Le Gouis et al., 2020). However, *Fusarium* head blight (FHB) - a devastating disease primarily caused by the fungal pathogen *Fusarium graminearum* - contributes to dramatic post-harvest losses and mycotoxin contamination in wheat, posing a threat to food security and safety worldwide (Goswami and Kistler,

2004). *F. graminearum* has the ability of producing type B trichothecenes (TCTB), such as deoxynivalenol (DON) and its acetylated derivatives (e. g., 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON)) (Tan et al., 2021), which are of high concern for human and animal health (EFSA Panel on Contaminants in the Food Chain (CONTAM) et al., 2017). In this sense, seeking the European Green Deal-oriented sustainable and eco-friendly strategies for managing FHB is crucial not only in protecting wheat plants but also in minimizing the overuse of agrochemicals.

In this study, we aim to shed light on the potential of stilbenoids

<footnote><sup>a</sup> INRAE, RU1264 Mycology and Food Safety (MycSA), 71, Avenue Edouard Bourlaux, Villenave d'Ornon 33883, France</footnote>

<footnote><sup>b</sup> Univ. Bordeaux, Bordeaux INP, INRAE, OENO, UMR 1366, ISVV, Villenave d'Ornon F-33140, France</footnote>

<footnote><sup>c</sup> Bordeaux Sciences Agro, Gradignan F-33170, France</footnote>

<footnote><sup>d</sup> Bordeaux Metabolome, MetaboHUB, PHENOME-EMPHASIS, Centre INRAE de Nouvelle Aquitaine-Bordeaux, Villenave d'Ornon F-33140, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: Trang.MinhTran@ugent.be (T. Tran-Minh), nadia.ponts@inrae.fr (N. Ponts), stephane.bernillon@inrae.fr (S. Bernillon), Pierre.Waffo-Teguo@ubordeaux.fr (P. Waffo-Téguo), Caroline.Rouger@u-bordeaux.fr (C. Rouger), florence.forget@inrae.fr (F. Richard-Forget), vessela.atanasova@inrae.fr (V. Atanasova).</footnote>

<footnote n="1">¹ Applied Mycology and Phenomics, Department of Plants and Crops, Ghent University, Valentin Vaerwyckweg 1, Ghent 9000, Belgium.</footnote>

<footnote n="2">² Department of Plant Biotechnology and Bioinformatics, Ghent University, K.L. Ledeganckstraat 35, Ghent 9000, Belgium.</footnote>
extracted from grapevine by-products as potential biofungicides that may significantly reduce the occurrence of FHB disease. In France, it is estimated that around 1.8 million tons of viticultural wood waste is generated annually (Zwingelstein et al., 2020). Numerous studies have demonstrated that vineyard-derived wastes are natural reservoirs of stilbenoids, a well-known class of phenolic compounds (Guerrero et al., 2016; Billet et al., 2021). Among these molecules, monomeric stilbenoids, e.g., resveratrol (RES), are predominantly found in the upper parts of the grapevine (e.g., grape canes), while oligomers, particularly vitisin B (VIT) - a tetramer of RES - are highly accumulated in the roots (e.g., rootstocks) (Gabaston et al., 2019; Tran et al., 2023; Tardif et al., 2024). Both RES and VIT typically exist in their E-isomer form rather than the Z-isomer in nature (Riviere et al., 2012). Prior studies have shown that these molecules exhibit potent antifungal and antimycotoxin properties against plant pathogens, as summarized in a review by Tran et al. (2023). More recently, a report by Tardif et al. (2024) revealed an abundance of stilbenoid oligomers showing fungicidal activity against F. graminearum in the rootstocks of five different French grape varieties. Several mechanisms have been proposed to explain the fungicidal activity of stilbenoids, including disruption of fungal cell walls and phospholipid membranes, as well as the inhibition of mycotoxin biosynthesis pathways (Tran et al., 2023) since DON serves as a virulent factor during infection of wheat plants by F. graminearum (Tan et al., 2020). Besides, to efficiently manage FHB, it is essential to comprehensively understand the DON biosynthesis pathways in F. graminearum. The enzymes involved in DON biosynthesis are encoded by 15 TRI genes, located in three distinct loci: a core TRI gene cluster containing 12 genes (TRI3 to TRI14), a two-gene locus (TRI1-TRI16), and a single gene locus TRI101, distributed across different chromosomes (Merhej et al., 2011a). Among these TRI genes, TRI6 and TRI10 function as transcriptional factors, while TRI5, encoding a trichodiene synthase, initiates biosynthesis by converting farnesyl diphosphate (FPP) into trichodiene. The TRI8-encoded esterase in particular has a dual function, catalysing deacetylation of 3,15-diacetyledeoxynivalenol at either carbon 15 or carbon 3, leading to the formation of 15-ADON and 3-ADON, respectively (Alexander et al., 2011). On the other hand, fungal cell wall and phospholipid bilayer of the plasma membrane, as the first points of contact with the host, play pivotal roles in fungal virulence (Rella et al., 2016; Zhang et al., 2019). It has been suggested that stilbenoids may disrupt these physical barriers by affecting the expression of genes involved in chitin and glucan biosynthesis, as well as those involved in sphingolipid metabolism (Tran et al., 2023). Their precise modes of action (MOA), however, remain unclear and require further exploration, which is the main objective of the present study.

To elucidate the aforementioned proposed molecular MOA, we challenged the fungus *F. graminearum* with RES and VIT - the two predominant stilbenoids extracted from vine by-products as reported by Tardif et al. (2024) - in liquid culture. We then studied the effects of these pure molecules on the fungal endo- and exo-metabolomes using untargeted liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS) and profiled transcriptomic changes via mRNA sequencing technology (mRNA-seq). Integrating these omics data could provide a comprehensive understanding of the mechanisms underlying the potent antifungal and antimycotoxin properties of these molecules. The insights gained from this study will support the transformation of undervalued grapevine wastes into stilbenoid-rich extracts with the potential for integration in new biofungicides that combat *F. graminearum*.

## 2. Materials and methodology

### 2.1. Preparation of materials

RES was purchased from TCI Europe (Zwijndrecht, Belgium), while VIT (purity > 95 % HPLC/DAD) was extracted and purified from grapevine by-products in the Molecules of Biological Interest laboratory

(ISVV, Villenave d'Ornon, France) following a procedure that has already been described elsewhere (Biais et al., 2017). Stock solutions of 7 mM RES and 1.6 mM VIT were prepared in 100 % ethanol (Thermo Fisher Scientific, Courtaboeuf, France) and kept at 4 °C. TCTB standards of DON and 15-ADON were purchased from Romer Labs (Tulln, Austria).

The *F. graminearum* CBS 185.32 strain (Westerdijk Institute, the Netherlands) classified as a DON/15-ADON chemotype (Atanasova-Penichon et al., 2018) was used throughout this study. To prepare spore solutions, the fungus was first cultured in a potato dextrose agar (PDA) (Difco, France) plate at 25 °C in darkness for seven days. Production of the fungal conidia was induced by transferring four fungal agar plugs (φ 0.5 cm) into 20 mL carboxymethyl cellulose (CMC) liquid medium (15 g L⁻¹ CMC, 1 g L⁻¹ yeast extract, 0.5 g L⁻¹ MgSO₄·7H₂O, 1 g L⁻¹ NH₄NO₃, and 1 g L⁻¹ KH₂PO₄) incubated at 25 °C and 180 rpm in a Multitron incubator shaker (INFORS AG, Bottmingen, Switzerland) for three days. Conidia were collected by filtering through a 100 µM Sefar Nitex membrane 03–100 (SEFAR AG, Heiden, Switzerland) followed by centrifugation at 4816 g for 10 min. The conidial pellet was resuspended and diluted in sterile water to a final concentration of 10⁶ conidia mL⁻¹. A synthetic mycotoxin medium (MS), as described by Boutigny et al. (2009), was used to promote TCTB production by *F. graminearum*.

### 2.2. Antifungal and antimycotoxin assay

#### 2.2.1. *In vitro* assays

*F. graminearum* was cultivated in submerged synthetic MS medium (Boutigny et al., 2009), which has been established as a suitable system for studying fungal development and mycotoxin accumulation. This medium promotes TCTB mycotoxin production and ensures close contact between fungal cells and bioactive molecules, making it appropriate for evaluating the molecular mechanisms of antifungal and antimycotoxin compounds (Atanasova-Penichon et al., 2018). *In vitro* antifungal and antimycotoxin effects of RES and VIT against *F. graminearum* CBS 185.32 were evaluated using six-well plates; each well contained 5 mL of MS medium supplemented with either RES (35 µM), VIT (8 µM), or 0.5 % ethanol as a negative control. Each well was inoculated with 100 µL of a conidial suspension of *F. graminearum* (10⁶ conidia mL⁻¹) and incubated at 25 °C in darkness. Production of fungal biomass and TCTB was quantified at 3, 5, 7, 10, and 14 days post-inoculation (dpi). At each time point, biomass was harvested by centrifugation (4816 g for 10 min) followed by lyophilization (48 h, Flexi-Dry, Oerlikon Leybold, Germany), while the supernatant was stored at -20 °C for TCTB and metabolomics analyses. Biomass harvested at 3 and 5 dpi was kept at -80 °C for RNA extraction. Four replicates were performed for each condition.

#### 2.2.2. Detached leaf assays

The effect of VIT was evaluated using detached leaf assays, as described by Tan et al. (2021). In brief, leaf segments (6 cm) were detached from 10-day-old soft wheat seedlings. Each segment was wounded in the central with a sterile 10 µL pipette tip prior to conveying onto water agar (5 g L⁻¹) square petri dishes amended with 40 g L⁻¹ benzimidazole. Five µL of *F. graminearum* spore suspension was deposited in the centre of each leaf segment followed by 5 µL of VIT (40 µM). Ethanol 0.5 % was used as a control. The disease progress was monitored within seven days by imaging with a digital camera at 4 and 7 dpi. The lesion area was measured using the ImageJ software package developed by the National Institutes of Health (Bethesda, Maryland, USA).

### 2.3. Quantification of type B trichothecenes by UHPLC-DAD

TCTB were extracted from culture media using ethyl acetate. Four mL of each supernatant was agitated with 8 mL of ethyl acetate (VWR, Fontenay-Sous-Bois, France) in a 15 mL PP tube for 3 min. Five mL of the upper organic phase was transferred into a glass tube prior to
evaporating at 45°C under nitrogen flux. Dried samples were dissolved in 200 µL of methanol/water (1:1, v/v) and filtered through a 0.2-µm PTFE Acrodisc mini-spike syringe filter. The filtrate was transferred into a micro-injection vial. TCTB yield was analysed using a Shimadzu Prominence ultra-high-performance liquid chromatography coupled with diode array detection (UHPLC-DAD) (Shimadzu Scientific Instruments, Noisiel, France). A Kinetex XB-C18 100 Å column (4.6 mm×150 mm, 2.6 µM) (Phenomenex, Le Pecq, France) maintained at 45 °C was used for separation. Water and acetonitrile were used as mobile phases A and B respectively. The following gradient elution setup was applied: 7%-30% B for 6 min; 30%-90% B for 2 min; 90% B for 2 min; 90%-7% B for 1 min; and 7% B for 6 min with a flow rate of 0.7 mL min⁻¹. UV spectra were recorded from 190 to 400 nm and peak areas were measured at 230 nm. Quantification of TCTB was conducted with an external calibration curve of DON and 15-ADON ranging from 2.5 to 500 µg mL⁻¹.

## 2.4. RNA extraction and library preparation for mRNA-seq

Total RNA was extracted from 30 mg *F. graminearum* frozen fresh biomass harvested at 3 and 5 dpi using TRIzol reagent (Thermo Fisher Scientific, USA) following the manufacturer's instructions. These time-points were chosen based on preliminary growth kinetics and trichothecene accumulation profiles, which indicate that key shifts in fungal metabolism and TCTB production occur between 48 and 96 h post-inoculation under *in vitro* conditions (*Atanasova-Penichon* et al., 2018; *Merhej* et al., 2011b; *Seong* et al., 2009). Quantification of RNA concentration was carried out using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific, USA), while the quality of RNA was examined on a 1.5 % agarose gel. RNA samples were diluted to a final concentration of 2 ng µl⁻¹ and stored at -80 °C until further analyses had been undertaken. Fifty µL of each RNA sample was used for library preparation following the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, USA) according to the manufacturer's protocols. In brief, poly(A) mRNA from total RNA was isolated using the NEBNext Poly(A) mRNA magnetic isolation module (NEB #E7490, New England BioLabs, USA). The purified mRNA was fragmented in a first-strand synthesis reaction buffer and random primer mix (2X) provided by the manufacturer at 94 °C for 15 min. The fragmented mRNA further proceeded to first-strand cDNA synthesis followed by second-strand cDNA synthesis. After purification, the double strand cDNA fragments underwent an end prep of the cDNA library prior to ligating with adaptors. The adaptor-ligated DNA was purified and enriched by PCR reaction. The quality of the prepared library was assessed using an Agilent TapeStation system (Agilent, USA) before being injected into the Illumina HiSeq 2000 sequencing system.

## 2.5. mRNA-seq analyses

The quality of raw paired-end reads (43 bp) was assessed using FastQC tool (Andrews, 2010). Low-quality reads and adapter sequences were removed using the Trimmomatic (v0.39) tool (Bolger et al., 2014), using the Truseq3-Pe-Neb.fa:2:30:7:5:true settings, along with the parameters HEADCROP:10 and MINLEN:30. The filtered reads were subsequently mapped to the reference genome using the STAR tool (v2.7.11a) (Dobin et al., 2013). The reference genome of *F. graminearum* PH-1 was obtained from Ensembl Fungi (ASM24013v3). BAM files generated by STAR were used for the featureCounts program (Liao et al., 2014) to quantify gene expression.

Following this, the raw counts obtained from featureCounts were used for differentially expressed gene (DEG) analysis with the DESeq2 (Love et al., 2014) and SARTools (Varet et al., 2016) packages in R. Features with low counts were discarded using independent filtering for each comparison. Normalization of read counts was carried out to correct the systematic technical biases. DEG analysis was conducted by comparing the treated and the control conditions by time point, with a

controlled false discovery rate (FDR) threshold of 0.05 used to identify statistically significant DEGs (Table S1 and S2). Log₂ fold changes (Log₂FC) and Benjamini-Hochberg-adjusted (BH-adjusted) p-values were calculated for ranking DEGs.

To identify functional categories and metabolic pathways, gene ontology (GO) (Table S3) and KEGG enrichment analyses (Table S4) were performed using the FungiDB tool (Basenko et al., 2018). Additionally, co-expression network analyses were performed using the Markov cluster algorithm (MCL) implemented in Cytoscape software (version 3.10.0) to identify gene clusters and explore potential regulatory relationships among DEGs.

## 2.6. Untargeted metabolomics using LC-HR-MSMS

LC-HR-MSMS analyses were performed on supernatants and methanolic extracts of freeze-dried ground mycelia. Concerning mycelia, the powders (2–10 mg) were suspended with 1 mL of methanol-water (70:30, v/v), acidified with 0.1 % formic acid and containing 1.37 mM methyl vanillate (Sigma-Aldrich, Saint-Quentin-Fallavier, France) as an internal standard (IS), and were sonicated for 15 min in an ice bath. The extracts were centrifuged and filtered through 0.2 µm PTFE Acrodisc mini-spike syringe filters (VWR, Fontenay-Sous-Bois, France) in HPLC vials.

To study the supernatants, 500 µL were filtered through 0.2-µm PTFE Acrodisc mini-spike syringe filters and mixed with 500 µL of methanol-water (1:1, v/v) containing 2.74 mM methyl vanillate to obtain a final concentration in HPLC vial of 1.37 mM methyl vanillate as an IS. The samples were stored at -20 °C for further analysis. A Vanquish UHPLC system (Thermo Fisher Scientific, Courtaboeuf, France) was used to separate metabolites on a reversed-phase C18 column Hypersil GOLD aQ (100 × 2.1 mm, 1.9 µm) (Thermo Fisher Scientific, Courtaboeuf, France), using 0.1 % formic acid in water as solvent A, 0.1 % formic acid in acetonitrile as solvent B, and the following gradient: 0-1.4 min, 5 % B; 1.4-4.5 min, 5%-25 % B; 4.5-11 min, 25%-70 % B; 11-14 min, 70%-95 % B; 14-17.5 min, 95 % B; 17.5-18 min, 95%-5 % B; 18-23 min, 5 % B. The flow rate was set at 0.4 mL L⁻¹. The column temperature was set at 35 °C. The injection volume was 2 µL. Metabolites were detected with a Q Exactive Focus mass spectrometer. Electrospray ionization in positive mode with full scan and data-dependent MS² was used. The scan rate for ions was fixed at 3 Hz for an *m/z* range of 80–1100 and a resolving power of 70,000 at *m/z* 200. For the data-dependent MS² (ddMS²), the resolving power was 17,500 at *m/z* 200 with isolation window of 3.0 *m/z* and dynamic exclusion at 2.0 s. Nitrogen was used as the sheath, auxiliary, and collision gas. The main MS parameters were set as follows: sheath gas flow rate, 50 a.u.; auxiliary gas flow rate, 13 a. u.; sweep gas flow rate, 0 a.u.; spray voltage, 3.5 kV; capillary temperature, 320 °C; S-lens RF level 50 %; auxiliary gas heater temperature, 350°C. A solution containing 10 µL of each sample was used as a quality control (QC) and was injected each 10 injections. A blank extraction of non-inoculated samples at each time point was also injected to identify and remove signals from culture medium.

## 2.7. Data analysis of metabolomics

The UHPLC-HRMS/MS raw data were processed in an untargeted manner using the Compound Discoverer 3.3.2.31 software (Thermo Fisher Scientific™) and the workflow is described in the supplementary information (Supplementary Information S11). From the resulting data matrix, compounds were filtered with the following parameters: "Background" set to false; "Norm. Area" containing any value in any file; "Name" not blank; and "Name" not containing the term "similar". For the mycelial experiment, four outlier samples were removed: control replicate 1 at 3 dpi (C.3.1); control replicate 4 at 5 dpi (C.5.4); VIT replicate 4 at 5 dpi (VIT.5.4); and VIT replicate 4 at 7 dpi (VIT.7.4). The final supernatant data matrix contained 1155 variables and 60 samples (Supplementary Table S5) while the final mycelia matrices comprised
890 variables and 56 samples (Supplementary Table S5).

Subsequent statistical analyses were performed using MetaboAnalyst 6.0 (Pang et al., 2024). In brief, data normalization was carried out using square root transformation followed by Pareto scaling. Differentially accumulated metabolic features (DAMF) were determined by comparing the treated and the control conditions, using a false discovery rate (FDR) threshold of 0.05 and a fold change cut-off of 2.0, with group variance assumed to be equal. Log<sub>2</sub>FC and FDR adjusted p-values were calculated for ranking DAMF. Principal component analysis (PCA) was conducted using the *prcomp* function and visualized with the *ggplot2* package in R. Upset plots were generated using *UpSetR* package (Conway et al., 2017).

## 2.8. Expression of results and statistical analyses

Data for TCTB production, fungal biomass, and lesion area were reported as mean values ± standard deviation from five or six biological replications. The normality of the data distribution was assessed using the Shapiro-Wilk test. When data were normally distributed, one-way ANOVA followed by a post-hoc Tukey test were applied. In cases where normality was not met, the non-parametric Kruskal-Wallis test followed by a post-hoc Dunn's test was used at a significant level of *p* < 0.05 (* *p* < 0.05, ** *p* < 0.01, *** *p* < 0.001).

# 3. Results

## 3.1. Antifungal and antimycotoxin activities of stilbenoids derived from grapevine by-products combatting Fusarium graminearum

This current work studied the biological activities of two stilbenoid molecules – RES, a monomer, and VIT, a tetramer - which were selected because of their predominance in grapevine by-product extracts, as documented by Tran et al. (2023). Submerged MS medium was used as an *in vitro* model to investigate the effect of stilbenoid compounds. Although *F. graminearum* behaves differently in terms of growth and toxin production in liquid versus solid media, *in vitro* assays remain a vital preliminary approach to unravel molecular mechanisms before validation in *in planta* experiments. The concentrations of 8 µM for VIT and 35 µM for RES, which correspond to approximately 7 mg L<sup>-1</sup>, were used according to a preliminary study that has tested the effects of various VIT concentrations on fungal growth and TCTB production by *F. graminearum* CBS 185.32. Fig. 1 shows that at all the considered time points, 8 µM VIT significantly inhibited *F. graminearum*'s fungal growth and TCTB production, whereas no inhibition was observed for RES, despite being at the higher concentration of 35 µM. A significant reduction of biomass production induced by VIT was observed from 3 dpi onward when comparing with the control (Figs. 1A and 1D). At 5 dpi, biomass production was significantly reduced by VIT by 66 %

compared to the control (*p*-value = 0.028). Nonetheless, this effect of VIT declined at the later timepoints.

Regarding TCTB accumulation in the supernatants (Figs. 1B and 1C), toxins were quantified at 10 and 14 days post-inoculation. Before 10 dpi neither DON nor 15ADON were detected in the supernatants with our quantification method. Exposure to VIT, caused a significant reduction in the yield of DON and 15-ADON produced by *F. graminearum*. At 10 dpi, the levels of DON and 15-ADON in the VIT-treated culture medium were significantly lower than those in the untreated control. VIT remarkably inhibited the production of 15-ADON by approximately 78 % (*p*-value = 0.028) (Fig. 1C). Nonetheless, this inhibitory effect decreased to 20 % at 14 dpi. VIT also significantly inhibited DON production by more than 40 % at both time points when compared to the control (Fig. 1B). By contrast, RES showed no significant effect on TCTB production compared to the control at both time points.

We further evaluated the fungicidal effect of VIT using detached wheat leaf assays. Considering the transition from *in vitro* to *in situ* experimentation, the concentration of VIT tested was increased by a factor of five (40 µM). This adjustment was necessary because stilbenoids are photosensitive compounds, and *trans*-stilbenoids can be converted into *cis*-isoforms under fluorescent or ultraviolet (UV) light irradiation, which may reduce their antifungal activity (Latva-Mäenpää et al., 2021). Such photochemical instability in *planta* can lower the effective concentration available at the pathogen-plant interface, and therefore a higher applied dose is required to achieve biological activity. Fig. 2 illustrates a significant reduction in lesion area caused by *F. graminearum* when treated with 40 µM VIT (*p*-value = 0.004). At 4 dpi, VIT significantly reduced the infection level by 74 % compared to the control. This effect remained highly effective at 7 dpi, with a significant reduction of 69 % (*p*-value = 0.001). Additionally, leaves treated solely with VIT showed no necrosis (Fig. 2B), demonstrating the leaf *in situ* non-phytotoxic property of this molecule at the tested concentration.

In summary, our data highlighted the potential antifungal and antimycotoxin activity of VIT extracted from grapevine by-products against *F. graminearum*. This prompted us to further investigate its MOA.

## 3.2. Effects of RES and VIT on Fusarium graminearum transcriptome

To provide insights into the molecular MOA underlying the antifungal and antimycotoxin activities of the stilbenoid molecules, RNA-seq analyses were conducted in *F. graminearum* cultures treated with either VIT (8 µM), RES (35 µM), or ethanol 0.5 % at 3 and 5 dpi (shown in Fig. 1). Transcriptomic comparisons were carried out between VIT (or RES) treatments and the ethanol 0.5 % control. Of the 14,164 genes identified in the genome of *F. graminearum* PH-1 strain (King et al., 2015), transcriptomics analyses showed that approximately half of the gene (6991 genes) expressions were significantly modified by VIT

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Impact of resveratrol (RES, 35 µM) and vitisin B (VIT, 8 µM) on production of biomass (A), deoxynivalenol (DON) (B) and 15-acetyldeoxynivalenol (15-ADON) (C) of *Fusarium graminearum* at different time points. (D) Liquid culture of *F. graminearum* inoculated with either RES, VIT, or ethanol 0.5 % control at different time points. Different letters depict a statistically significant difference between treatments at each time point using a non-parametric Kruskal-Wallis test followed by a post-hoc Dunn test at a significance level of α= 0.05. dpi: days post inoculation.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Antifungal activity of vitisin B (VIT, 40 µM) against *Fusarium graminearum* using detached wheat leaf assay. (A) Lesion areas caused by *F. graminearum* were compared between treatments at four and seven days post inoculation (dpi). (B) Detached leaf assays were observed at 4 dpi. Leaves were either inoculated with pathogen (+Fg) ($10^6$ conidia mL$^{-1}$) or treated with phosphate-buffered saline (PBS) (-Fg) prior to treatment with either ethanol 0.5 % (control) or VIT. Asterisks indicate statistically significant differences between treatments, determined using a non-parametric Kruskal-Wallis test followed by a posthoc Dunn test at a significant level of $p < 0.05$ (* $p < 0.05$, ** $p < 0.01$, *** $p < 0.001$).</figcaption></figure>

(Fig. 3, Table S1 and S2). Strikingly, at both time points, wider transcriptomic regulations were induced by VIT rather than by RES, as shown in Figs. 3A and 3C. The PCA plots reveal a significant difference in transcriptomic regulation between VIT and the control group, as reflected by PC1 values of 95.29 % at 3 dpi and 91.21 % at 5 dpi. By contrast, the difference between RES and the control appeared as very slight with PC2 values of 3.2 % and 5.38 % at 3 and 5 dpi, respectively. Importantly, the three replicates per treatment clustered closely together, demonstrating the minimal variation within treatments. At 3 dpi, the Venn diagrams indicate that 6991 genes were significantly regulated by VIT, with 3475 genes downregulated and 3516 upregulated (Fig. 3D). At 5 dpi, 38 % of the genes downregulated at 3 dpi remained downregulated, while an additional 926 genes were uniquely downregulated compared to 3 dpi. Regarding upregulation, about one-third of the genes that were upregulated at 3 dpi remained upregulate at 5 dpi following exposure to VIT (Fig. 3D), while this molecule induced the upregulation of 964 other genes. By contrast, RES exposure was shown to affect the expression of only 1639 genes including 808 genes downregulated and 831 genes upregulated at 3 dpi. The effect of RES was remarkably reduced at 5 dpi. In fact, only 4 % of the genes regulated by RES at 3 dpi were shown to be modulated in their expression at 5 dpi. Volcano plots reported in Fig. 3B illustrate the stronger effects of VIT compared to RES on *F. graminearum* transcriptome.

### 3.2.1. GO enrichment analyses of DEGs

To provide deeper insights into the MOA of VIT, we performed functional enrichment analyses for DEGs in which *F. graminearum* was exposed to this molecule. Fig. 4A shows significant GO enrichment of the downregulated gene sets induced by VIT at 3 and 5 dpi (Table S3). At 3 dpi, 31 GO terms related to biological process (BP) and 18 related to cellular component (CC) were identified. At 5 dpi, there were 3 BP-related, 18 CC-related, and 1 molecular function (MF)-related GO terms. Strikingly, VIT significantly downregulated genes involved in an array of fungal lipid biosynthesis-related BPs, encompassing lipid metabolic process (GO:0006629), cellular lipid metabolic process (GO:0044255), phospholipid metabolic process (GO:0006644), lipid biosynthetic process (GO:0008610), membrane lipid biosynthetic process (GO:0046467), and fatty acid biosynthetic process (GO:0006633). Exposure to VIT resulted in the downregulation of BPs related to the development and reproduction of *F. graminearum*, including sporulation (GO:0043934), structure development (GO:0048856), structure morphogenesis (GO:0009653), reproductive process (GO:0022414), and cell differentiation (GO:0030154). Additionally, VIT negatively affected secondary metabolisms by significantly downregulating polyketide (GO:0030639), lactone (GO:1901334), zearalenone

(GO:0106150), and antibiotic (GO:0016999) metabolic processes. On the other hand, VIT demonstrated negative effects on components of the fungal membrane at both observed time points. Indeed, most of the enriched CC-related GO terms of the downregulated gene sets were related to membrane components, particularly intrinsic components of membrane (GO:0031224), endomembrane system (GO:0012505), and endoplasmic reticulum membrane (GO:0005789).

On the other hand, GO enrichment analyses of the upregulated gene sets identified 173 significantly enriched GO terms at 3 dpi and 207 significantly enriched GO terms at 5 dpi. Fig. 4B exhibits the top 10 GO terms with the highest fold enrichment by categories (BP, CC, and MF) and time points (Table S3). Remarkably, nucleobase-containing compound and amino acid biosynthesis process-related genes were significantly enriched, particularly those involved in inosine monophosphate (IMP) metabolic process (GO:0046040), purine nucleoside monophosphate biosynthetic process (GO:0009127), IMP biosynthetic process (GO:0006188), and branched-chain amino acid biosynthetic process (GO:0009082). We also found significant CC-related GO terms relevant to ribosomal components (Fig. 4B). Strikingly, VIT triggered the upregulation of genes related to the translation regulator activity (GO:0045182) and catalytic activity of RNA polymerase (GO:0003899), aminoacyl-tRNA ligase (GO:0004812), RNA helicase (GO:0003724), and RNA methyltransferase (GO:0008173).

### 3.2.2. KEGG enrichment analyses of DEGs

To identify significant metabolic pathways influenced by VIT and RES, Kyoto encyclopaedia of genes and genomes (KEGG) enrichment analyses of DEGs were performed (Fig. 5, Table S4). As illustrated in Fig. 5, data associated with VIT treatment evidenced a stronger impact on the metabolic pathways in *F. graminearum* compared to RES at both time points. At 3 dpi, both molecules significantly downregulated DEGs involved in ubiquinone and other terpenoid-quinone biosynthesis (KEGG:ec00130) with 110 DEGs for VIT vs. 40 DEGs for RES and sphingolipid metabolism (KEGG:ec00600), with 71 DEGs for VIT vs. 25 DEGs for RES. The VIT-induced downregulated DEGs were also significantly enriched in glycerophospholipid metabolism (KEGG:ec00564).

By contrast, KEGG enrichment analyses of the VIT-induced upregulated DEGs showed that VIT significantly upregulated 9 metabolic pathways at 3 dpi and eight at 5 dpi. Among the pathways affected by VIT at 3 dpi, it is worth mentioning the purine metabolism (KEGG:ec00230 with 145 DEGs), pyruvate metabolism (KEGG:ec00620 with 94 DEGs), amino acid metabolism (e.g., valine, leucine, and isoleucine metabolism (KEGG:ec00260 with 101 DEGs)), and aminoacyl-tRNA biosynthesis (KEGG:ec00970 with 54 DEGs). Furthermore, we found that VIT and RES molecules significantly triggered the citrate cycle in
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Globally transcriptomic regulation of *Fusarium graminearum* induced by vitisin B (VIT, 8 µM) and resveratrol (RES, 35 µM) compared to the untreated control at three and five days post inoculation (dpi). (A) Principal component analyses. (B) Volcano plots indicating differentially expressed genes (DEGs) by pairwise comparisons (VIT vs. the untreated control, RES vs. the untreated control, and VIT vs. RES) at different significant levels of false discovery rate (FDR)-adjusted p-values (i.e., FDR ≤ 0.05, FDR ≤ 0.01, and FDR ≤ 0.001). Each dot is representative of a DEG. Log₂ Fold Change (FC) > 0 correspond to the up-regulation of a gene, or else the down-regulation of a gene. (C) A heatmap of significantly DEGs upon treatment with either VIT or RES compared to the control. (D) Venn diagrams showing the distribution of differentially down- and up-regulated genes induced by VIT and RES. Darker blue or red colours indicate a higher number of DEGs.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Gene ontology (GO) enrichment analyses of *Fusarium graminearum* (A) down and (B) up-regulated differentially expressed genes (DEGs) induced by vitisin B (VIT). GO terms are significantly enriched at a Bonferroni-corrected p-value < 0.05 computed by Fisher's exact tests prior to log₁₀ transformation.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** KEGG enrichment analyses of *Fusarium graminearum* differentially expressed genes (DEGs) induced by resveratrol and vitisin B at three and five days post-inoculation (dpi). Significantly enriched metabolic pathways are identified based on Bonferroni-corrected *p*-values.</figcaption></figure>

F. graminearum (KEGG:ec00020 with 17 DEGs for RES at 3 dpi and 29 DEGs for VIT at 5 dpi).

**Fig. 6** shows the co-expression network analyses of 3580 VIT-induced significant DEGs with $|log_2FC| > 1$ at 3 dpi. Functional enrichment analyses were performed on the modules containing the most significantly down- or upregulated genes. Notably, VIT triggered

the downregulation of gene clusters associated with lipid biosynthesis pathways, particularly those involved in steroids (14 genes), long-chain fatty acids (5 genes), sphingolipids (5 genes), and carotenoid metabolism (9 genes). The co-expression analysis also identified a cluster of 8 genes related to aurofusarin metabolism that were co-downregulated by VIT.
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Co-expression network analyses of 3580 VIT-induced significant differentially expressed genes (DEGs) with $|log₂FC| > 1$ observed at three days post-inoculation. Each node represents an individual gene and clustering was performed using Markov cluster algorithm (MCL) in Cytoscape software (version 3.10.0). Functional enrichment analysis was applied to the modules with most downregulated genes shown in blue and upregulated shown in red (p-adj < 0.05). Edges represent functional relationships between the genes.</figcaption></figure>

### 3.3. Mode of action of stilbenoids against Fusarium graminearum

In this section, we propose a number of mechanisms by which VIT is highly effective, based on the aforementioned GO and KEGG enrichment analyses. These mechanisms include: (1) disruption of fungal cell wall and plasma membrane; (2) inhibition of fungal sporulation and structure formation; (3) downregulation of genes involved in mycotoxin biosynthesis pathway; and (4) other possible modes of action such as interference with fungal cell cycle, epigenetic effect, and regulation of apoptosis and autophagy. Accordingly, we comprehensively analysed the expression levels of DEGs involved in specific metabolic pathways and/or cellular physiological functions in *F. graminearum*.

#### 3.3.1. Disruption of fungal cell wall and plasma membrane

Fig. 7 shows the expression levels of DEGs induced by VIT and RES that are related to fungal cell walls and plasma membrane biosynthesis. VIT significantly downregulated key genes involved in the biosynthesis of three main components of fungal cell wall, including chitin (e.g. FGRAMPH1_01G12837 encoding chitin synthase 1, and FGRAMPH1_01G03149 encoding chitin synthase 4), glucan (e.g. FGRAMPH1_01G04885: 1,3-beta-glucanosyltransferase), and ergosterol (e.g. FGRAMPH1_01G06005: C-5 sterol desaturase) (Fig. 7A-7C) at both time points. Moreover, most of the genes involved in membrane lipid biosynthesis were significantly downregulated by VIT at both time points (Fig. 7D). Additionally, Fig. 7E highlighted four putative genes involved in sphingolipid metabolism that were significantly down-regulated by VIT, consisting of FGRAMPH1_01G26045 (Log₂FC = -1.0), FGRAMPH1_01G06053 (Log₂FC = -2.2), FGRAMPH1_01G07943 (Log₂FC = -1.5), and FGRAMPH1_01G13861 (Log₂FC = -5.4). FGRAMPH1_01G06053-encoding sphinganine C4-hydroxylase (SUR2), which catalyses the hydroxylation of sphinganine to phytosphingosine, was significantly suppressed by VIT compared to the untreated control.

#### 3.3.2. Inhibition of fungal sporulation and structure formation

To align with the GO terms enriched in sporulation (GO:0043934) and structure development (GO:0048856), we further investigated the expression of key genes involved in these processes. Fig. 8 illustrates a significant downregulation of such genes in *F. graminearum* when exposed to VIT at 3 dpi. Notably, the gene FGRAMPH1_01G07441, which encodes developmental regulatory protein wetA required for fungal

conidiation (Son et al., 2014), was significantly suppressed by VIT (Log₂FC = -2.8) (Figs. 8A and 8B). Conversely, no considerable effect was observed for RES. Moreover, our data have demonstrated the anti-germination activity of VIT against *F. graminearum* (Fig. S1).

#### 3.3.3. Downregulation of genes involved in mycotoxin biosynthesis pathway

VIT supplementation in *F. graminearum* liquid cultures led to strong inhibition of the production of 15-ADON and DON in *F. graminearum* (Fig. 1). As shown in Fig. 9A, this inhibition was associated with a downregulation of most of the genes involved the TCTB biosynthesis pathway at the early time points of 3 and 5 dpi. TRI10 and TRI6, the two master transcriptional regulators of TCTB biosynthesis in *F. graminearum*, were significantly suppressed by VIT (Fig. 9A). VIT also caused a significant reduction in the expression of other TRI genes, particularly TRI5 which encodes the trichodiene synthase - the first enzyme in the TCTB pathway. By contrast, RES, the less active molecule, did not lead to such a transcriptional control.

On the other hand, VIT significantly downregulated the expression of genes involved in other polyketide biosynthesis pathways (Fig. 9B). Notably, VIT considerably reduced the expression of FGRAMPH1-01G05593 (polyketide synthase 12 (PKS12)) and FGRAMPH1_01G05603 (aurS), the two key genes responsible for the biosynthesis of aurofusarin, at both time points. Significant down-regulation induced by VIT was also observed for genes encoding non-ribosomal peptide synthetases, especially FGRAMPH1_01G21959 (NRPS14) and FGRAMPH1_01G09373 (NRPS7).

### 3.4. Effects of RES and VIT on secondary metabolism of F. graminearum

The effects of VIT and RES on the endo-metabolome (metabolites in mycelia) and exo-metabolome (metabolites in supernatants) of *F. graminearum* at different developmental stages were studied using untargeted LC-HR-MSMS metabolomic analysis. PCA were performed in order to evaluate whether metabolomic profiles discriminate between treated and control conditions (Fig. 10). The PCA score plots show that the first two components account for 69 % of the total variance in mycelia and 84 % in supernatants. At all time points, RES-treated and control samples are grouped together for both the mycelia and supernatants, suggesting no significant differences in their metabolomic profiles. By contrast, VIT-treated samples were separated from control
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Expression profiles of *differentially expressed genes* (DEGs) set induced by vitisin B (VIT) and resveratrol (RES) involved in (A) chitin, (B) glucan, (C) ergosterol, (D) membrane lipid biosynthesis, and (E) sphingolipid biosynthesis pathway. Expression levels are presented as log~2FC~.</figcaption></figure>

samples at all time points except for 14 dpi.

Accordingly, we provided insights into the effect of VIT on the endo- and exo-metabolomes of *F. graminearum* by identifying significantly differentially abundant metabolic features in both mycelia and supernatants, compared to the control. As illustrated on the upset plot reported in Fig. 11 and Table S6, VIT significantly affected the secondary metabolism of *F. graminearum*. Regarding mycelial analyses, VIT negatively influenced the intracellular metabolism of the fungus, with 861 significantly differentially reduced metabolic features (DRMFs) identified at 3 dpi, 119 at 5 dpi, 86 at 7 dpi, and 119 at 10 dpi (Fig. 11B), compared to the control. The effect was less pronounced at 14 dpi, with only 10 DRMFs detected. Notably, the intersection showed that 76 significantly DRMFs were detected at 3, 5, and 7 dpi. On the other hand, exposure to VIT induced the accumulation of several metabolic features.

For example, we found 48 significantly differentially accumulated metabolic features (DAMFs) in VIT-treated mycelia at 5 dpi (Fig. 11A), compared to the control.

The data shown in Fig. 11D also revealed that VIT had a negative impact on the extracellular metabolism of *F. graminearum*. At 3 dpi, 684 significantly DRMFs were detected in the supernatant of VIT-treated cultures (Fig. 11B), compared to the control. This amount was 195, 344, and 388 DRMFs at 5, 7, and 10 dpi, respectively. Remarkably, 81 significantly DRMFs were intersected at three timepoints of 3, 5, and 7 days. At 14 dpi only 11 significantly DRMFs were found. Nonetheless, VIT also significantly triggered production of several extracellular secondary metabolites. As shown in Fig. 11C, 52 DAMFs were found in the supernatant at 10 dpi and 16 DAMFs at 14 dpi.

In summary, the data demonstrated that VIT had a significantly
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Expression profiles of differentially expressed genes (DEGs) involved in the fungal sporulation (A) and structure formation (B) induced by vitisin B (VIT) and resveratrol (RES). Expression levels are presented as log<sub>2</sub>FC.</figcaption></figure>

negative impact on the extra- and endo-metabolism of *F. graminearum*. The bioactivity of this molecule was most pronounced at the early time points.

### 3.4.1. TCTB biosynthesis pathway

Next, we specifically assessed the effect of VIT on the TCTB biosynthesis pathway in *F. graminearum* (Fig. 12). Differential metabolomic analysis (DMA) showed that VIT had a pronounced impact on this secondary metabolic pathway. At 3 dpi, VIT significantly downregulated 50 and 183 putative DAMs associated with TCTB biosynthesis pathways (shown in Fig. 12C) in mycelia and supernatant, respectively (Table S7 and S8). In mycelia the reduced metabolic features consisted of trichodiene, 2-hydroxytrichodiene, trichotriol, isotrichodiol, trichodermol, isotrichodermol, isotrichodermin, and 15-acetyl deoxynivalenol (Fig. 12A). In the supernatant, the significantly reduced metabolites were trichodiene, 2-hydroxy trichodiene, 12,13-epoxy-9,10-trichoene-2-ol, 12,13-epoxytrichothec-9-ene, isotrichodiol, trichotriol,

trichodermol, isotrichodermin, 15-decalonectrin, calonectrin, 7,8-dihydroxy calonectrin, 3,15-acetyl deoxynivalenol, 15-acetyl deoxynivalenol (15-ADON), and deoxynivalenol (DON) (Fig. 12B). Notably, VIT led to a significant reduction in the production of 15-ADON in both the mycelium (Log<sub>2</sub>FC = -2.4) and supernatant (Log<sub>2</sub>FC = -3.2) at 3 dpi. However, the effect of VIT diminished over time, as illustrated by its nonsignificant impact at 14 dpi. By contrast, RES had no pronounced impact on the TCTB metabolism in *F. graminearum*.

### 3.4.2. Sphingolipid metabolism

The aforementioned GO and KEGG enrichment analyses demonstrated that VIT significantly downregulated lipid metabolic process, particularly sphingolipid metabolism, in *F. graminearum*. This finding was further supported by the observation that metabolites involved in the sphingolipid biosynthesis pathway (Fig. 7E) were remarkably less abundant in VIT-treated cultures. This was observed at 3 dpi, at which the yield of sphinganine, a key intermediate, along with its derivatives,
<figure><img src="image_13.png" /><figcaption>(B) Polyketide biosynthesis pathways</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Fig. 9.** Expression profiles of differentially expressed genes (DEGs) involved in trichothecene type B (TCTB) (A) and polyketide (B) biosynthesis pathways induced by vitisin B (VIT) and resveratrol (RES) at three and five days post-inoculation (dpi). Expression levels are presented as log~2~FC.</figcaption></figure>

was suppressed by VIT (Fig. 13).

4. Discussion

Natural stilbenoids derived from agro-forest wastes exhibit potent fungicidal activities against a broad spectrum of fungal pathogens, as documented by Tran et al. (2023). More recently, by integrating

high-resolution mass spectrometry (HRMS) data with the biological activities of hydroethanolic extracts from French grapevine by-products through untargeted metabolomic approaches, Tardif et al. (2024) reported that the oligomers of stilbenoids were major actors of the anti-fungal and antimycotoxin activities of extracts (Tardif et al., 2024). Among the studied extracts, the more active, Merlot and Tannat root extracts, completely suppressed fungal growth when 125 mg L⁻¹ of the
<figure><img src="image_15.png" /><figcaption>**Fig. 10.** Principle component analyses of *Fusarium graminearum* secondary metabolism in (A) mycelia (56 samples, 890 variables) and (B) supernatant (60 samples, 1155 variables) induced by vitisin B (VIT) and resveratrol (RES) compared to the control (C). Untargeted metabolomic analyses were conducted on mycelia and supernatant samples at 3, 5, 7, 10, and 14 days.</figcaption></figure>

extract was supplemented to the liquid medium and, at a concentration of 25 mg L⁻¹, reduced mycotoxin production by 84% and 98%, respectively (Tardif et al., 2024). Metabolic profiles of these extracts showed that stilbenoid oligomers, particularly VIT, were predominant. This molecule represents 72% and 58% of total quantified stilbenoids, respectively, suggesting that it could play a central role in the extract's bioactivity. To test this hypothesis, we evaluated the effects of VIT - a tetramer of RES - at 8 µM and RES - the "flagship" molecule of stilbenoids for the formation of numerous derivatives - at 35 µM. We found that VIT exhibited significantly stronger effects against *F. graminearum* compared to RES. While VIT inhibited both fungal growth and TCTB production considerably, RES showed no significant effects (Fig. 1). In detached leaf assays, VIT (at 40 µM) significantly reduced the disease severity caused by *F. graminearum*, leading to a profoundly smaller lesion compared to the control (Fig. 2).

Following this, we aimed to gain a comprehensive understanding of the MOA associated with the antifungal and antimycotoxin properties of VIT and RES. Both transcriptomics and metabolomics were used, with the data showing that at all analysed time points of fungal development, the samples that had been treated with RES were clustered with the control samples; this finding suggests that this compound does not induce significant modifications in *Fusarium* metabolism (Figs. 3A, 10A, and 10B). These results, which corroborate the lack of effect on vegetative growth and TCTB production, are consistent with the findings of previous studies in which no or only minimal effects of RES were observed on *F. graminearum*, *Fusarium oxysporum*, *Fusarium verticillioides*, and *Aspergillus carbonarius* (Marin et al., 2006; Crespo-Sempere et al., 2016; Song et al., 2021). Previous studies that reported RES's antifungal activity, however, used much higher concentrations of RES than the

present study, i.e., 1920 µM against *B. cinerea* (Xu et al., 2018) and 876 µM against *F. verticillioides* and *F. graminearum* (Marin et al., 2006). Only one study targeting *Aspergillus flavus* witnessed any antifungal activity on the part of RES at a concentration close to that used in this study, i.e., 13.1 µM (Wang et al., 2015), suggesting that the effects of RES vary depending on fungal species. By contrast, VIT was shown to induce significant modifications in both primary and secondary fungal metabolisms. Yet, these modifications gradually decreased over the course of the culture, becoming negligible at 14 dpi. This may be due to the low concentration of VIT used (8 µM), which is more than 60 times lower than that of other phenolic compounds studied, such as ferulic and caffeic acids (Yan et al., 2023), as well as to fungal adaptive responses and chemical degradation of the compound. One possible mechanism is the upregulation of fungal efflux pumps, such as ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters, which can actively export toxic compounds like stilbenoids, reducing their intracellular concentrations and mitigating their antifungal effects (Del Sorbo et al., 2000). Additionally, *F. graminearum* may induce the expression of detoxification enzymes such as laccases, which are known to metabolize or inactivate phenolic compounds. Research has posited that laccase enzymes secreted by the fungus (Frandsen et al., 2006) may be responsible for the biotransformation of stilbenoid molecules. Taillis et al. (2023), for example, reported that Botrytis laccase possesses a strong capacity to transform and/or degrade certain stilbenoids such as RES and r2-viniferin (i.e., a tetramer, synonymous with vitisin A) (Taillis et al., 2023). However, the previous authors found that r-viniferin (syn. vitisin B) had not been metabolized by *B. cinerea* secretome five hours after inoculation, suggesting that the biotransformation of stilbenoids might vary between species, medium, and incubation time. On the other
<figure><img src="image_16.png" /><figcaption>**Fig. 11.** Upset plots illustrate the significantly differentially abundant metabolic features identified in mycelia and supernatant induced by vitisin B (VIT) compared to the untreated control (C). Plots A and B present the accumulated and reduced metabolic features in mycelia respectively. Plots C and D depict the accumulated and reduced metabolic features in supernatant respectively. The set size represents the total number of metabolic features detected at each time point, while the intersection size indicates the number of metabolic features detected across multiple timepoints.</figcaption></figure>

hand, VIT degradation could occur independently of fungal activity, as this compound is chemically unstable and prone to isomerization and oxidation over time, particularly under light or aerobic conditions (Latva-Mäenpää et al., 2021). These mechanisms, alone or in combination, may explain the observed time-dependent decline in VIT activity against *F. graminearum*.

With regard to the antifungal MOA of VIT, our metabo-
transcriptomic analysis indicates that VIT could serve as a chitin/
glucan synthesis inhibitor in the early stages of fungal development,
leading to the disruption of the fungal cell wall. As shown in Figs. 7A and
7B, this molecule disrupts the first physical barrier via suppressing the
expression of genes involved in chitin and glucan biosynthesis. Chitin
synthases play critical roles in both the vegetative development and
virulence of *F. graminearum* (Kim et al., 2009; Liu et al., 2016). A study
by Niu et al. (2020) illustrated that (7E)-2,4-dihydroxy-1-methylstilbene
(48.6 µM) reduced the fungal growth by 50 % by possibly targeting the
cell wall of *F. graminearum* (Niu et al., 2020). Additionally, VIT signifi-
cantly downregulated the sphingolipid metabolisms (Figs. 7C, 7D, 7E
and 13), potentially compromising the integrity of the phospholipid
bi-layer. Notably, we found that VIT significantly suppressed the

expression of *SUR2* (Figs. 7D and 7E). It has been demonstrated that
knocking out *SUR2* increased the higher susceptibility of *F. graminearum*
to azole fungicides (Wang et al., 2023). Likewise, using a transmission
electron microscope, prior studies found that other stilbenoids, e.g.,
pterostilbene (975 µM) (Xu et al., 2022) and resveratrol (263 µM)
(Adrian and Jeandet, 2012), were capable of impairing the plasma
membrane integrity of *Botrytis cinerea*. Electron microscopy revealed
that these compounds inhibit *B. cinerea* by altering the morphology of
the hyphae and conidiophores, with destruction of the plasma mem-
brane and organelles. We further confirmed the anti-germination ac-
tivity of VIT, as shown in Figure S1. On the other hand, sporulation is a
well-known and essential biological process in the life cycle of the
*Fusarium* species (Ajmal et al., 2022), with the *wetA* gene playing a
central regulatory role. In particular, the deletion of *wetA* has been
shown to increase *F. graminearum*'s susceptibility to various stresses
(Son et al., 2014). In this study, we found that VIT treatment led to the
downregulation of *wetA*, suggesting a potential mechanism for its
anti-sporulation activity.

In addition, VIT significantly impacted other pathways in the pri-
mary and secondary metabolisms tightly linked to the TCTB pathway,
<figure><img src="image_17.png" /><figcaption>**Fig. 12.** Profiles of differentially abundant metabolic features putatively linked to the trichothecene type B (TCTB) pathways in *Fusarium graminearum*, detected in biomass (A) and supernatant (B) after exposure to vitisin B (VIT) and resveratrol (RES) at various time points. Putatively metabolic features that were significantly accumulated or reduced were identified based on log₂FC values. (C) TCTB biosynthesis pathways adapted from metacyc.org, highlighting key putative metabolites and TRI genes. Metabolites shown in red represent key mycotoxins produced by *F. graminearum*. TRI genes highlighted in purple were significantly downregulated by VIT. DPI: days post-inoculation.</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>**Fig. 13.** Profiles of significantly differentially abundant metabolic features putatively linked to the Sphingolipid biosynthesis pathway in *Fusarium graminearum*, detected in mycelium after exposure to vitisin B (VIT) and resveratrol (RES) at various time points. Significantly reduced and accumulated metabolic features are identified based on log₂FC values. DPI: days post inoculation.</figcaption></figure>

such as the TCA cycle, pyruvate metabolism, and polyketide biosynthesis pathway. To counteract the VIT-induced stress, we hypothesized that the fungus might alter its primary metabolism particularly through upregulation of the TCA cycle and pyruvate metabolism (Fig. 5 and S2A&B). All of these pathways share the same precursor, the acetyl-CoA, with polyketide biosynthesis. Upregulation of TCA cycle genes could suggest that the fungus increased ATP demand for stress response mechanisms, such as detoxification and repair processes, resulting in less available acetyl-CoA for the biosynthesis of the secondary pathways such as sesquiterpene (including TCTB) and polyketide pathways. Consistent with this, the fungus upregulated oxidative stress response

genes, particularly glutathione S-transferase (GSTs), as shown in Figure S3. GSTs have been implicated in the detoxification of carbendazim, a commonly used fungicide, in *F. graminearum* (Sevastos et al., 2017). While caffeic acid has been shown to promote the production of several polyketides by inhibiting TCTB biosynthesis (Atanasova-Penichon et al., 2018), VIT, however, significantly downregulated the expression of *PKS12*, *aurS*, *FGRAMPH1_01G05589* responsible for the biosynthesis of the polyketides aurofusarin and its precursor, rubrofusarin, as well as zearalenone (Fig. 4). With regard to pyruvate metabolism, Han et al. (2025) have suggested that abiotic stress - in the form of heat treatment - was responsible for an increase in
intracellular pyruvate content, contributing to the improvement of heat
resistance of *Ganoderma lucidum* (Han et al., 2025). On the other hand,
VIT induced the upregulation of purine metabolism (Fig. S2C) which
plays a vital role in vegetative growth and development in
*F. graminearum* (Sun et al., 2021). It is assumed that the fungus upre-
gulated DNA/RNA synthesis in response to VIT-induced stress.

Furthermore, both omics datasets demonstrated that VIT signifi-
cantly reduced the production of TCTB by hampering the expression of
*TRI* genes at the early time points (Figs. 9 and 12). This result was
consistent with targeted metabolomics data shown in Fig. 1. Notably,
the yields of most intermediate metabolites for the production of 15-
ADON and DON were significantly reduced by VIT at 3 days
(Figs. 12A and 12B). This was due to the significant downregulation of
the genes that encode the first steps of TCTB biosynthesis pathway, i.e.,
*TRI4*, *TRI5*, and *TRI11* (Fig. 12C). In addition, the expression of two
positive transcription factors, *TRI6* and *TRI10*, were downregulated in
comparison with the control, while *TRI101* and *TRI8* showed the
opposite expression. The *TRI6* gene was also reported as the main
regulator of the TCTB biosynthesis.

Last but not least, the transcriptomics showed that VIT might alter
the fungal cell cycle and the epigenetic effect while regulating fungal
apoptosis and autophagy (Fig. S4). Evidently, exposure to VIT induced a
significant downregulation of a gene encoding probable septin *aspE*
(Fig. S4A). Prior studies have shown molecular functions of septin
protein in cytokinesis, asexual and sexual development, lipid meta-
bolism, nuclear division, morphogenesis, and virulence in *Fusarium*
(Chen et al., 2016; Zhang et al., 2017). VIT also regulated the fungal
epigenetics by downregulating genes that encode histone, H4, H3, H2A.
Z, and H2A - core components of the nucleosome - as well as the
histone-lysine N-methyltransferase *set9* (Fig. S4B). This downregulation
may disrupt chromatin structure and alter gene expression, thereby
contributing to the reduced fungal growth and TCTB production in
*F. graminearum* (Gu et al., 2017; Hao et al., 2023). Intriguingly, VIT
appeared to induce apoptosis - programmed cell death - in
*F. graminearum*, as evidenced by the upregulation of a caspase-like
protein (*FGRAMPH1_01G16451*), particularly from the C14 family pro-
teinases (Fig. S4D) (Kabbage, Williams, and Dickman, 2013) which may
contribute to its antifungal activity. In response to VIT-induced stress,
the fungus may activate autophagy as a compensatory survival mecha-
nism, shown in the upregulation of autophagy-related genes 3, 5, 8, 17,
and 18 (Fig. S4C) (Kabbage, Williams, and Dickman, 2013). However,
the precise functional roles of these genes in *F. graminearum* still require
further investigation.

5. Conclusion

These findings enhance our understanding of the MOA of stilbenoids
and highlight the potential to recycle agricultural waste, such as vine by-
products, as a source of novel and sustainable strategies for managing
Fusarium head blight and mycotoxin contamination in wheat.

This study presents a comprehensive comparative transcriptomic
and metabolomic analysis of *F. graminearum*'s response to two stilbe-
noids extracted from vine by-products and revealed that exposure to VIT
significantly affects key fungal metabolic pathways. Building on the
insights gained from this biomolecule, further research is needed to
better understand the MOA of the crude stilbenoid extract derived from
grapevine by-products, which represents a more practical and accessible
option for field applications. In addition, the observed antifungal and
antimycotoxin activities of the extract cannot be attributed to a single
active molecule. Rather, they likely result from synergistic interactions
among multiple compounds. Moreover, other constituents whether
acting independently or in synergy with stilbenoids are also likely to
contribute to the overall bioactivity of the extract.

In the short term, the stability of the active crude extract under two
critical environmental factors, temperature and UV exposure, should be
thoroughly investigated to support the development of an optimized

formulation. Furthermore, to improve the extract’s bioefficacy, and
enhance its surface activity, the incorporation of selected technical ad-
juvants commonly used in the plant protection and spraying industry
may be considered.

From an application perspective, grapevine by-product extracts are especially promising, as they are cost-effective to produce at large scale and expected to display stronger efficacy due to natural synergy among metabolites. Future work will therefore focus on validating these extracts in *in planta* assays and field trials. Purified VIT will serve as a benchmark reference to confirm efficacy and to anchor mechanistic insights, ensuring a translational bridge between laboratory findings and field applications.

CRediT authorship contribution statement

**Vessela Atanasova:** Writing – review & editing, Validation, Resources, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization. **Stéphane Bernillon:** Writing – review & editing, Validation, Methodology. **Pierre Waffo-Téguo:** Writing – review & editing, Resources, Project administration, Funding acquisition. **Caroline Rouger:** Writing – review & editing. **Florence Richard-Forget:** Writing – review & editing, Validation, Supervision, Resources, Methodology, Conceptualization. **Trang Tran-Minh:** Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. **Nadia Ponts:** Validation, Supervision, Methodology, Data curation.

Funding sources

This project was financially supported by the French Agence Natio-
nale de la Recherche through the StilDeTox (ANR- 19-CE21-0007)
project.

Declaration of Competing Interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgements

Authors thank The Language Room France for English review of the
manuscript.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.micres.2025.128370.

Data availability

Raw RNA-seq data have been deposited in the European Nucleotide
Archive (ENA) under the assessment number PRJEB91901. These data are
currently private but accessible to reviewers if required.

References

Adrian, M., Jeandet, P., 2012. Effects of resveratrol on the ultrastructure of *botrytis cinerea* conidia and biological significance in plant/pathogen interactions. *Fitoterapia* 83 (8), 1345–1350. https://doi.org/10.1016/j.fitote.2012.04.004.

Ajmal, M., Hussain, A., Ali, A., Chen, H., Lin, H., 2022. Strategies for controlling the sporulation in Fusarium spp. J. Fungus 9 (1), 10. https://doi.org/10.3390/jof9010010.

Alexander, N.J., McCormick, S.P., Waalwijk, C., Van der Lee, T., Proctor, R.H., 2011. The genetic basis for 3-ADON and 15-ADON trichothecene chemotypes in *Fusarium*. *Fungal Genet. Biol.* 48 (5), 485–495. https://doi.org/10.1016/j.fgb.2011.01.003.
Andrews, S. FastQC: a Quality Control Tool for High Throughput Sequence Data. Cambridge, United Kingdom; 2010. (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

Atanasova-Penichon, L., Legoaec, L., Bernillon, S., Deborde, C., Maucourt, M., Verdal-Bonnin, M.-N., Pinson-Gadais, L., Ponts, N., Moing, A., Richard-Forget, F., 2018. Mycotoxin biosynthesis and central metabolism are two interlinked pathways in *Fusarium graminearum*, as demonstrated by the extensive metabolic changes induced by caffeic acid exposure. Appl. Environ. Microbio 84 (8), e01705-17. https://doi.org/10.1128/AEM.01705-17.

Basenko, E.Y., Pulman, J.A., Shanmugasundram, A., Harb, O.S., Crouch, K., Starns, D., Warrenfeltz, S., Aurrecochea, C., Stoeckert, Jr.C.J., Kissinger, J.C., 2018. FungiDB: an integrated bioinformatic resource for fungi and oomycetes. J. Fungus 4 (1), 39. https://doi.org/10.3390/jof4010039.

Biais, B., Krisa, S., Cluzet, S., Da Costa, G., Waffo-Teguo, P., Mérillon, J.-M., Richard, T., 2017. Antioxidant and cytoprotective activities of grapevine stilbenes. J. Agric. Food Chem. 65 (24), 4952-4960. https://doi.org/10.1021/acs.jafc.7b01254.

Billet, K., Unlubayir, M., Munsch, T., Malinowska, M.A., De Bernonville, T.D., Oudin, A., Courdavault, V., Besseau, S., Giglioli-Guivarc'h, N., Lanoue, A., 2021. Postharvest treatment of wood biomass from a large collection of european grape varieties: impact on the selection of polyphenol-rich byproducts. Chem. Eng. 9 (9), 3509-3517. https://doi.org/10.1021/acssuschemeng.0c07875.

Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 30 (15), 2114-2120. https://doi.org/10.1093/bioinformatics/btu170.

Boutigny, A.-L., Barreau, C., Atanasova-Penichon, L., Verdal-Bonnin, M.-N., Pinson-Gadais, L., Richard-Forget, F., 2009. Ferulic acid, an efficient inhibitor of type b trichothecene biosynthesis and tri gene expression in fusarium liquid cultures. Mycol. Res. 113 (6-7), 746-753. https://doi.org/10.1016/j.mycres.2009.02.010.

Chen, A., Xie, Q., Lin, Y., Xu, H., Shang, W., Zhang, J., Zhang, D., Zheng, W., Li, G., Wang, Z., 2016. Septins are involved in nuclear division, morphogenesis and pathogenicity in *Fusarium graminearum*. Fungal Genet. Biol. 94, 79-87. https://doi.org/10.1016/j.fgb.2016.07.005.

Conway, J.R., Lex, A., Gehlenborg, N., 2017. UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics 33 (18), 2938-2940. https://doi.org/10.1093/bioinformatics/btx364.

Crespo-Sempere, A., Selma-Lázaro, C., Palumbo, J.D., González-Candelas, L., Martínez-Culebras, P.V., 2016. Effect of oxidant stressors and phenolic antioxidants on the ochratoxigenic fungus *Aspergillus carbonarius*. J. Sci. Food Agric. 96 (1), 169-177. https://doi.org/10.1002/jfsa.7077.

Del Sorbo, G., Schoonbeek, H., De Waard, M.A., 2000. Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal Genet. Biol. 30, 1-15. https://doi.org/10.1006/fgbi.2000.1206.

Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., Gingeras, T.R., 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29 (1), 15-21. https://doi.org/10.1093/bioinformatics/bts635.

EFSA Panel on Contaminants in the Food Chain (CONTAM), Knutsen, H.K., Alexander, J., Grasl-Kraupp, B., Hogstrand, C., Hoogenboom, L. (Ron), Nebbia, C.S., Oswald, I.P., Petersen, A., Rose, M., Roudot, A.-C., Schwertdle, T., Vlembeckx, C., Vollmer, G., Wallace, H., De Saeger, S., Eriksen, G.S., Farmer, P., Fremy, J.-M., Gong, Y.Y., Meyer, K., Naegeli, H., Parent-Massin, D., Rietjens, I., van Egmond, H., Altieri, A., Eskola, M., Gergelova, P., Ramos Bordajandi, L., Benkova, B., Dörr, B., Gkrillers, A., Gustavsson, N., van Manen, M., Edler, L., 2017. Risks to human and animal health related to the presence of deoxynivalenol and its acetylated and modified forms in food and feed. EFSA Journal 15, e04718. https://doi.org/10.2903/j.efsa.2017.4718.

EUROSTAT. Agricultural Production - Crops. 2024. (https://ec.europa.eu/eurostat/statistics-explained/index.php?title=Agricultural_production_crops#Cereals).

Frandsen, R.J., Nielsen, N.J., Maolanon, N., Sørensen, J.C., Olsson, S., Nielsen, J., Giese, H., 2006. The biosynthetic pathway for auroferusin in *Fusarium graminearum* reveals a close link between the naphthalinones and naphthopyrones. Mol. Microbiol. 61 (4), 1069-1080. https://doi.org/10.1111/j.1365-2958.2006.05295.x.

Gabaston, J., Leborgne, C., Waffo-Teguo, P., Valls, J., Palos Pinto, A., Richard, T., Cluzet, S., Merillon, J.M., 2019. Wood and roots of major grapevine cultivars and rootstocks: a comparative analysis of stilbenes by UHPLC-DAD-MS/MS and NMR. Phytochem. Anal. 30 (3), 320-331. https://doi.org/10.1002/pca.2815.

Goswami, R.S., Kistler, H.C., 2004. Heading for disaster: *Fusarium graminearum* on cereal crops. Mol. Plant Pathol. 5 (6), 515-525. https://doi.org/10.1111/j.1364-3737.2004.00252.x.

Gu, Q., Ji, T., Sun, X., Huang, H., Zhang, H., Lu, X., Wu, L., Huo, R., Wu, H., Gao, X., 2017. Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in *Fusarium verticillioides*. FEMS Microbiol. Lett. 364 (19), fnx184. https://doi.org/10.1093/femsle/fnx184.

Guerrero, R.F., Biais, B., Richard, T., Puertas, B., Waffo-Teguo, P., Merillon, J.-M., Cantos-Villar, E., 2016. Grapevine cane's waste is a source of bioactive stilbenes. Ind. Crop Prod. 94, 884-892. https://doi.org/10.1016/j.indcrop.2016.09.055.

Han, J., Tang, X., Wang, L., Chen, H., Liu, R., Zhao, M., 2025. GISIRT1 deacetylates and activates pyruvate kinase to improve pyruvate content and enhance heat stress resistance in *Ganoderma lucidum*. Microbiol. Res. 293, 128055. https://doi.org/10.1016/j.micres.2025.128055.

Hao, L., Zhang, M., Yang, C., Pan, X., Wu, X., Lin, H., Ma, D., Yao, Y., Fu, W., Chang, J., 2023. The epigenetic regulator Set9 harmonizes fungal development, secondary metabolism, and colonization capacity of *Aspergillus flavus*. Int. J. Food Microbiol 403, 110298. https://doi.org/10.1016/j.ijfoodmicro.2023.110298.

Kabbage, M., Williams, B., Dickman, M.B., 2013. Cell death control: the interplay of apoptosis and autophagy in the pathogenicity of *Sclerotinia sclerotiorum*. PLoS Pathog. 9 (4), e1003287. https://doi.org/10.1371/journal.ppat.1003287.

Kim, J.-E., Lee, H.-J., Lee, J., Kim, K.W., Yun, S.-H., Shim, W.-B., Lee, Y.-W., 2009. *gibberella zeae* chitin synthase genes, GzCHS5 and GzCHS7, are required for hyphal growth, perithecia formation, and pathogenicity. Curr. Genet 55, 449-459. https://doi.org/10.1007/s00294-009-0258-6.

King, R., Urban, M., Hammond-Kosack, M.C., Hassani-Pak, K., Hammond-Kosack, K.E., 2015. The completed genome sequence of the pathogenic ascomycete fungus *Fusarium graminearum*. BMC Genom. 16, 1-21. https://doi.org/10.1186/s12864-015-1756-1.

Latva-Mäenpää, H., Wufu, R., Mulat, D., Sarjala, T., Saranpää, P., Wöhlä, K., 2021. Stability and photoisomerization of stilbenes isolated from the bark of Norway spruce roots. Molecules 26 (4), 1036. https://doi.org/10.3390/molecules26041036.

Le Gouis, J., Oury, F.-X., Charmet, G., 2020. How changes in climate and agricultural practices influenced wheat production in Western Europe. J. Cereal Sci. 93, 102960. https://doi.org/10.1016/j.jcs.2020.102960.

Liao, Y., Smyth, G.K., Shi, W., 2014. Featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30 (7), 923-930. https://doi.org/10.1093/bioinformatics/btt656.

Liu, Z., Zhang, X., Liu, X., Fu, C., Han, X., Yin, Y., Ma, Z., 2016. The chitin synthase FgChs2 and other FgChs co-regulate vegetative development and virulence in *F. graminearum*. Sci. Rep. 6 (1), 34975. https://doi.org/10.1038/srep34975.

Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 1-21. https://doi.org/10.1186/s13059-014-0550-8.

Marin, S., Ramos, A., Cuevas, D., Sanchis, V., 2006. *Fusarium verticillioides* and *Fusarium graminearum* infection and fumonisin B1 and zearalenone accumulation in resveratrol-treated corn. Food Sci. Technol. Int. 12 (4), 353-359. https://doi.org/10.1177/1082013206067.

Merhej, J., Richard-Forget, F., Barreau, C., 2011a. Regulation of trichothecene biosynthesis in *Fusarium*: recent advances and new insights. Appl. Microbiol. Biotechnol. 91, 519-528. https://doi.org/10.1007/s00253-011-3397-x.

Merhej, J., Richard-Forget, F., Barreau, C., 2011b. The ph regulatory factor Pac1 regulates tri gene expression and trichothecene production in *Fusarium graminearum*. Fungal Genet. Biol. 48, 275-284. https://doi.org/10.1016/j.fgb.2010.11.008.

Niu, S., Liu, T., Deng, Y., Wang, W., Zhang, Y., Hong, W., Zhang, D., Hua, J., Luo, S., 2020. Production and evaluation of antifungal stilbenoids in *Dracaena cochinchinensis* elicited by fungal inoculation. Ind. Crop Prod. 145, 112148. https://doi.org/10.1016/j.indcrop.2020.112148.

Pang, Z., Lu, Y., Zhou, G., Hui, F., Xu, L., Viau, C., Spigelman, A.F., MacDonald, P.E., Wishart, D.S., Li, S., 2024. MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Res. 52 (W1), W398-W406. https://doi.org/10.1093/nar/gkae253.

Rella, A., Farnoud, A.M., Del Poeta, M., 2016. Plasma membrane lipids and their role in fungal virulence. Prog. Lipid Res. 61, 63-72. https://doi.org/10.1016/j.plipres.2015.11.003.

Riviere, C., Pawlus, A.D., Merillon, J.-M., 2012. Natural stilbenoids: distribution in the plant kingdom and chemotaxonomic interest in *Vitaceae*. Nat. Prod. Rep. 29 (11), 1317-1333. https://doi.org/10.1039/C2NP20049J.

Seong, K.Y., Pasquali, M., Zhou, X., Song, J., Hilburn, K., McCormick, S., Dong, Y., Xu, J., Kistler, H.C., 2009. Global gene regulation by *Fusarium* transcription factors Tri3 and Tri10 reveals adaptations for toxin biosynthesis. Mol. Microbiol. 72, 354-367. https://doi.org/10.1111/j.1365-2958.2009.06649.x.

Sevastos, A., Labrou, N., Flouri, F., Malandrakis, A., 2017. Glutathione transferase-mediated benzimidazole-resistance in *Fusarium graminearum*. Pestic. Biochem. Physiol. 141, 23-28. https://doi.org/10.1016/j pestbp.2016.11.002.

Son, H., Kim, M.-G., Min, K., Lim, J.Y., Choi, G.J., Kim, J.-C., Chae, S.-K., Lee, Y.-W., 2014. WetA is required for conidiogenesis and conidium maturation in the ascomycete fungus *Fusarium graminearum*. Eukaryot. Cell 13 (1), 87-98. https://doi.org/10.1128/ec.00220-13.

Song, P., Yu, X., Yang, W., Wang, Q., 2021. Natural phytoalexin stilbene compound resveratrol and its derivatives as anti-tobacco mosaic virus and anti-phytopathogenic fungus agents. Sci. Rep. 11 (1), 16509. https://doi.org/10.1038/s41598-021-96069-1.

Sun, M., Bian, Z., Luan, Q., Chen, Y., Wang, W., Dong, Y., Chen, L., Hao, C., Xu, J.R., Liu, H., 2021. Stage-specific regulation of purine metabolism during infectious growth and sexual reproduction in *Fusarium graminearum*. New Phytol. 230 (2), 757-773. https://doi.org/10.1111/nph.17170.

Taillis, D., Becissa, O., Pébarthé-Courrouilh, A., Renouf, E., Palos-Pinto, A., Richard, T., Cluzet, S., 2023. Antifungal activities of a grapevine byproduct extract enriched in complex stilbenes and stilbenes metabolization by *Botrytis cinerea*. J. Agric. Food Chem. 71 (11), 4488-4497. https://doi.org/10.1021/acs.jafc.2c07843.

Tan, J., Ameye, M., Landschoot, S., De Zutter, N., De Saeger, S., De Boevre, M., Abdallah, M.F., Van der Lee, T., Waalwijk, C., Audenaert, K., 2020. At the scene of the crime: new insights into the role of weakly pathogenic members of the fusarium head blight disease complex. Mol. Plant Pathol. 21 (12), 1559-1572. https://doi.org/10.1111/mpp.12996.

Tan, J., De Zutter, N., De Saeger, S., De Boevre, M., Tran, T.M., Van der Lee, T., Waalwijk, C., Willems, A., Vandamme, P., Ameye, M., 2021. Presence of the weakly pathogenic *Fusarium poae* in the fusarium head blight disease complex hampers biocontrol and chemical control of the virulent *Fusarium graminearum* pathogen. Front. Plant Sci. 12, 641890. https://doi.org/10.3389/fpls.2021.641890.

Tardif, C., Rouger, C., Miranda, J., Ahmed, O.S., Richard-Forget, F., Atanasova, V., Waffo-Teguo, P., 2024. Targeting of antifungal metabolites from grapevine byproducts by UPLC-HRMS/MS approaches using bioactivity-based molecular
networking. J. Agric. Food Chem. 72 (17), 9621-9636. https://doi.org/10.1021/acs.jafc.3c09531.

Tran, T.M., Atanasova, V., Tardif, C., Richard-Forget, F., 2023. Stilbenoids as promising natural product-based solutions in a race against mycotoxigenic fungi: a comprehensive review. J. Agric. Food Chem. 71 (13), 5075-5092. https://doi.org/10.1021/acs.jafc.3c00407.

Varet, H., Brillet-Guéguen, L., Coppée, J.-Y., Dillies, M.-A., 2016. SARTools: a DESeq2-and EdgeR-based r pipeline for comprehensive differential analysis of RNA-Seq data. PLoS One 11 (6), e0157022. https://doi.org/10.1371/journal.pone.0157022.

Wang, H., Lei, Y., Yan, L., Cheng, K., Dai, X., Wan, L., Guo, W., Cheng, L., Liao, B., 2015. Deep sequencing analysis of transcriptomes in *Aspergillus flavus* in response to resveratrol. BMC Microbiol. 15, 1-14. https://doi.org/10.1186/s12866-015-0513-6.

Wang, H., Zhang, Y., Wang, J., Chen, Y., Hou, T., Zhao, Y., Ma, Z., 2023. The sphinganine C4-hydroxylase FgSur2 regulates sensitivity to azole antifungal agents and virulence of *Fusarium graminearum*. Microbiol. Res. 271, 127347. https://doi.org/10.1016/j.micres.2023.127347.

Xu, D., Deng, Y., Han, T., Jiang, L., Xi, P., Wang, Q., Jiang, Z., Gao, L., 2018. in vitro and in vivo effectiveness of phenolic compounds for the control of postharvest gray mold of table grapes. Postharvest Biol. Technol. 139, 106-114. https://doi.org/10.1016/j.postharvbio.2017.08.019.

Xu, D., Qiao, F., Xi, P., Lin, Z., Jiang, Z., Romanazzi, G., Gao, L., 2022. Efficacy of pterostilbene suppression of postharvest gray mold in table grapes and potential mechanisms. Postharvest Biol. Technol. 183, 111745. https://doi.org/10.1016/j.postharvbio.2021.111745.

Yan, H., Meng, X., Lin, X., Duan, N., Wang, Z., Wu, S., 2023. Antifungal activity and inhibitory mechanisms of ferulic acid against the growth of *Fusarium graminearum*. Food Biosci. 52, 102414. https://doi.org/10.1016/j.fbio.2023.102414.

Zhang, Y.-Z., Chen, Q., Liu, C.-H., Lei, L., Li, Y., Zhao, K., Wei, M.-Q., Guo, Z.-R., Wang, Y., Xu, B.-J., 2019. *fusarium graminearum* FgCWM1 encodes a cell wall mannoprotein conferring sensitivity to salicylic acid and virulence to wheat. Toxins 11 (11), 628. https://doi.org/10.3390/toxins11110628.

Zhang, Y., Gao, T., Shao, W., Zheng, Z., Zhou, M., Chen, C., 2017. The septins FaCdc3 and FaCdc12 are required for cytokinesis and affect asexual and sexual development, lipid metabolism and virulence in *Fusarium asiaticum*. Mol. Plant Pathol. 18 (9), 1282-1294. https://doi.org/10.1111/mpp.12492.

Zwingelstein, M., Draye, M., Besombes, J.-L., Piot, C., Chatel, G., 2020. Viticultural wood waste as a source of polyphenols of interest: opportunities and perspectives through conventional and emerging extraction methods. Waste Manag. 102, 782-794. https://doi.org/10.1016/j.wasman.2019.11.034.