Fusarium species are among the most economically damaging pathogens of wheat, barley, maize and other cereals worldwide. They cause seedling blight, crown and root rot, and the devastating head blight of wheat and barley, and they contaminate grain with mycotoxins such as deoxynivalenol and other trichothecenes that threaten food and feed safety. Managing these fungi has relied heavily on triazole-based fungicides and crop rotation, yet chemical control is imperfect, and meta-analyses of triazole efficacy against Fusarium head blight and deoxynivalenol accumulation show only partial protection. Resistance to fungicides within Fusarium populations is an increasing concern, and regulatory pressure on agrochemicals continues to grow. Against this backdrop, plant-derived phenolic compounds have attracted attention as possible biopesticides, since plants themselves deploy these molecules as chemical defenses against fungal invaders.

The research team, led by Farah Hamici of the University of Bouira in Algeria, together with colleagues from Bouira and Jendouba University in Tunisia, set out to answer two linked questions: how the choice of extraction technology and solvent shapes the phytochemical profile of the two plants, and whether the resulting extracts differ in their ability to suppress Fusarium growth. The researchers compared three environmentally friendly extraction techniques: conventional maceration, ultrasound-assisted extraction, which uses acoustic cavitation to rupture plant cell walls, and microwave-assisted extraction, which heats intracellular water rapidly to burst cells from within. Each technique was performed with two solvents, distilled water and a 70 percent ethanol solution, giving a matrix of six extraction conditions per plant.

The quantitative results demonstrated that both the method and the solvent significantly influenced phytochemical recovery. Microwave-assisted extraction generally delivered the highest total phenolic contents, consistent with the idea that rapid internal heating liberates phenolics more completely than slower techniques. Ultrasound-assisted extraction with the hydroethanolic solvent, by contrast, proved especially effective for flavonoids, a subclass of polyphenols that includes many well-known antifungal and antioxidant agents. The authors note that this kind of method-dependent variation matters practically, because a bioactivity observed in one extract may vanish in another prepared from the same plant with a slightly different protocol. Standardizing extraction is therefore a prerequisite for any serious attempt to turn these plants into reproducible crop-protection products.

Antioxidant performance was assessed with two standard radical-scavenging assays, DPPH and ABTS, which measure how effectively an extract neutralizes synthetic free radicals. Ruta chalepensis emerged as the stronger and more consistent antioxidant of the two species. Its DPPH half-maximal inhibitory concentrations, or IC50 values, ranged from 3.5 to 4.7 micrograms per milliliter, remarkably low figures that indicate very potent radical scavenging, while ABTS values ranged from 4.0 to 13.8 micrograms per milliliter. Fraxinus angustifolia, whose manna and bark have long been valued in Mediterranean traditional medicine and whose phenolic composition has been characterized in earlier work, showed antioxidant activity as well, though less consistently across the extraction conditions.

The antifungal assays formed the heart of the study. The team tested the extracts in vitro against two cereal-associated pathogens, Fusarium redolens and Fusarium culmorum, using methods adapted from standardized microtiter plate monitoring protocols for Fusarium growth. The results pointed clearly in one direction: Fusarium redolens was the more susceptible of the two species, and the hydroethanolic extracts of Ruta chalepensis were the most inhibitory preparations overall. Minimum inhibitory concentrations, or MIC values, for the most active rue extracts fell below 50 micrograms per milliliter against Fusarium redolens, an impressively low threshold for a crude plant extract, and ranged from 79.34 to 82.38 micrograms per milliliter against the more tolerant Fusarium culmorum. Extracts of Fraxinus angustifolia also inhibited both fungi, but generally required higher concentrations.

To understand which molecules might be responsible, the researchers subjected the most active extracts to HPLC-DAD analysis, a chromatographic technique that separates compounds and detects them by their absorption of ultraviolet and visible light. Among the compounds showing the largest relative peak areas in the most active preparations were quercetin and a dihydroxyflavone. Quercetin in particular has an extensive literature behind it: studies of its antimicrobial mechanisms describe multiple modes of action, including disruption of fungal membrane integrity, inhibition of energy metabolism and interference with cell wall biosynthesis. The presence of these flavonoids at high relative abundance in the extracts that performed best against the fungi suggests, though it does not yet prove, that they contribute substantially to the observed antifungal effect.

An important caveat, and one that the broader literature on natural products emphasizes, is that crude plant extracts are not single drugs but complex mixtures whose components may interact synergistically or antagonistically. Researchers studying natural product extracts have repeatedly shown that whole extracts can outperform any isolated constituent, because multiple compounds attack multiple fungal targets simultaneously, making resistance harder to evolve. The Algerian team’s finding that hydroethanolic rue extracts, rich in several phenolics at once, were the most fungistatic preparations is consistent with such a multi-component mode of action. It also echoes earlier reports of antifungal activity in Ruta species, including work on rue essential oils from the Moroccan High Atlas and on enriched extracts of Ruta graveolens against other Fusarium species.

The choice of Fusarium redolens as a test organism gives the study particular significance. This soil-borne species has been flagged in recent reviews as an emerging plant pathogen, notably in Iran, where it has been implicated in cereal root rots, yet it remains understudied relative to its headline-grabbing relatives. Because no previous study had evaluated macerated, ultrasound-assisted or microwave-assisted extracts of either rue or narrow-leafed ash against this fungus, the low MIC values reported here establish a genuine first. They also suggest that phenolic-rich extracts could be integrated into management strategies for crown and root rot diseases, which are receiving renewed attention across North African wheat-growing regions as climate variability alters disease pressure.

Several steps remain before any of this translates into a field-ready biopesticide. In vitro inhibition of mycelial growth does not guarantee efficacy on seeds or in soil, where extract stability, rainfastness, phytotoxicity to the crop and effects on beneficial soil microbes all come into play. Whether the extracts also suppress mycotoxin production, not just fungal growth, is a separate and critical question, since phenolic compounds have been shown in some systems to reduce trichothecene biosynthesis and in others to have no such effect. Formulation, scale-up of extraction and cost analysis will determine whether microwave- or ultrasound-assisted hydroethanolic extraction of rue leaves is economically viable at agricultural scale. Nevertheless, the study offers a compelling proof of concept: two plants with deep roots in Mediterranean folk medicine, extracted with green technologies and simple solvents, produce preparations that stop a cereal pathogen at concentrations measured in tens of micrograms per milliliter. As fungicide resistance spreads and consumers demand lower chemical residues in food, such plant-based defenses are moving from ethnobotanical curiosity toward practical crop protection, and this first look at rue and ash against Fusarium redolens suggests they deserve a place in that conversation.

Subject of Research: Antifungal activity of phytochemical extracts from Ruta chalepensis and Fraxinus angustifolia against cereal Fusarium pathogens

Article Title: Phytochemical characterization and antifungal activity of Fraxinus angustifolia and Ruta chalepensis against cereal Fusarium species: first evaluation against Fusarium redolens

Article References: Phytochemical characterization and antifungal activity of Fraxinus angustifolia and Ruta chalepensis against cereal Fusarium species: first evaluation against Fusarium redolens. (n.d.). https://doi.org/10.1007/s44473-026-00246-x

Image Credits: AI Generated

DOI: 10.1007/s44473-026-00246-x

Keywords: Ruta chalepensis, Fraxinus angustifolia, Fusarium redolens, Fusarium culmorum, antifungal activity, plant extracts, phenolic compounds, quercetin, microwave-assisted extraction, ultrasound-assisted extraction, biopesticides, cereal pathogens

The study, published in the journal Stress Biology, is notable for its scale and rigor. While previous investigations of barley’s response to Fusarium have largely focused on a single molecular layer, most often gene expression, this work integrated three unbiased approaches on the very same plant material. The team grew four barley varieties, Avalon, Barke, Morex and Palmella Blue, which differ in their susceptibility to initial infection but all share barley’s characteristic type II resistance, which prevents the fungus from spreading through the rachis of the ear. The researchers sprayed the spikes with F. culmorum spores around mid-flowering and sampled the tissue four and seven days later.

To confirm that infection had actually taken hold, the team quantified fungal DNA relative to barley DNA using quantitative PCR. The results tracked known differences in susceptibility: the resistant varieties Avalon and Barke carried the lowest fungal loads at seven days post inoculation, at 4.39 and 5.56 picograms of fungal DNA per nanogram of barley DNA respectively, while the more susceptible Palmella Blue accumulated 22.38 picograms. This gradient of infection success gave the researchers a meaningful backdrop against which to interpret the molecular data.

On the transcript level, the team used 3′-RNA sequencing mapped to the Morex V3 reference genome and detected transcripts from 36,920 gene models. Differential expression analysis identified 3,085 genes whose activity changed after infection in at least one variety at one time point. The proteomics side was equally ambitious: using an untargeted, bottom-up approach with tandem mass tag labeling and high-resolution mass spectrometry, the researchers quantified 7,671 proteins, of which 3,169 showed significantly altered abundance after infection. This shotgun proteomics dataset represents a major advance over earlier barley FHB studies, which relied on lower-depth two-dimensional gel methods.

The crucial analytical step came from overlaying the two datasets. The researchers identified 7,149 gene-protein pairs for which both the transcript and the corresponding protein had been measured, and within these they pinpointed 570 pairs that were significantly regulated at both the RNA and protein levels. These differentially expressed and differentially abundant pairs represent the most reliable candidates for genuinely deployed defense machinery, because they show evidence of both transcriptional activation and successful translation into protein. Enrichment analysis of this subset pointed unambiguously toward aromatic amino acid metabolism: terms and pathways related to the shikimate pathway, tryptophan biosynthesis, phenylalanine biosynthesis and phenylpropanoid biosynthesis were all significantly overrepresented.

Among the most strongly induced genes and proteins were the canonical players of plant immunity. Pathogenesis-related proteins, including chitinases, thaumatin-like proteins and PR1, showed dramatic increases, with log2-fold transcript changes reaching nearly 11 in some cases. The team also detected strong upregulation of known Fusarium response factors, including UDP-glycosyltransferases such as HvUGT13248, which detoxifies the mycotoxin deoxynivalenol by attaching a glucose group, and glutathione-S-transferases. But the single most consistently and strongly upregulated proteins across all varieties and time points were tryptophan decarboxylases, the enzymes that convert tryptophan into tryptamine, a precursor of serotonin.

The metabolomics data brought the story full circle. Out of more than 14,000 metabolic features detected, the researchers identified 53 unique metabolites at varying confidence levels, including tryptophan derivatives, hydroxycinnamic acids, hydroxycinnamic acid amides and the barley-specific hordatines. Serotonin and tryptamine were among the metabolites most strongly enriched in infected tissue, matching the enzyme data. Using an in silico database of known and hypothetical hydroxycinnamic acid amides, the team additionally identified conjugates linking hydroxycinnamic acids to agmatine, tryptamine and serotonin. Several of these, including caffeoyl-tryptamine and cinnamoyl-serotonin, accumulated after infection, while others decreased, possibly because they were being incorporated into the cell wall and thus became inaccessible to extraction.

The hordatines deserve special attention. These dimerized hydroxycinnamoylagmatine conjugates are found only in barley and its wild relatives, and they have a documented history as antifungal compounds. Hordatine A inhibits spore germination of several fungi, including Fusarium oxysporum, at concentrations as low as ten micromolar in liquid culture. In the new study, coumaroylagmatine, the biosynthetic precursor, along with hordatine A and its glucoside, all rose after infection. The agmatine coumaroyltransferase genes that initiate this branch of the pathway were highly upregulated at the transcript level in every variety, echoing earlier findings in wheat and Brachypodium where loss or silencing of equivalent enzymes increases susceptibility to Fusarium.

What makes the convergence so compelling is that all three omics layers, measured independently on the same samples, point to the same metabolic hub. The shikimate pathway funnels carbon into chorismate, which feeds both tryptophan and phenylalanine biosynthesis. Tryptophan yields tryptamine and serotonin; phenylalanine yields hydroxycinnamic acids; and the two streams merge in the hydroxycinnamic acid amides, including the hordatines. Because all four barley varieties, despite their differences in type I resistance to initial infection, share type II resistance to fungal spread, the authors hypothesize that this aromatic amino acid-derived chemical defense may contribute to barley’s natural ability to contain the pathogen within initially infected spikelets, a trait wheat lacks.

The study also flagged intriguing questions for future work. Nearly 2,000 proteins changed in abundance without any corresponding change in their transcripts, hinting at post-translational regulation or toxin-driven inhibition of translation by deoxynivalenol, which blocks the ribosome. Small proteins like the Fusarium resistance orphan gene HvFROG, whose transcript surged more than eleven-fold in one variety, escaped proteomic detection entirely, underscoring the limits of untargeted workflows. The researchers suggest that follow-up experiments with higher spatial and temporal resolution, and functional genetic validation of hydroxycinnamic acid amide metabolism, will be needed to confirm whether these compounds are genuinely causal for barley’s type II resistance. If they are, the aromatic amino acid pathways identified here could offer breeders chemical markers and genetic targets for building more resilient cereal varieties at a time when Fusarium Head Blight remains a persistent global threat to food safety and supply.

Subject of Research: Multi-omics analysis of barley defense metabolism during Fusarium Head Blight infection

Article Title: Multi-omics of barley Fusarium Head Blight converge on pathogen-triggered biosynthesis of aromatic amino acid derived chemical defense compounds

Article References: Hein, S., Steidele, C. E., Hoheneder, F., Brajkovic, S., Kuster, B., Kurzweil, L., Stark, T. D., Dawid, C., & Hückelhoven, R. (2026). Multi-omics of barley Fusarium Head Blight converge on pathogen-triggered biosynthesis of aromatic amino acid derived chemical defense compounds. Stress Biology, 6(1), Article 41. https://doi.org/10.1007/s44154-026-00313-5

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00313-5

Keywords: Fusarium Head Blight, barley, Fusarium culmorum, multi-omics, proteomics, metabolomics, tryptophan metabolism, serotonin, hordatines, hydroxycinnamic acid amides, plant immunity, mycotoxin detoxification