Wheat is a crucial cereal crop in the Triticum genus, and it serves as a dietary staple for nearly 40% of the world's population (Iqbal et al., 2022). Despite being the second most crucial staple after rice (Singh et al., 2016; Özdemir, 2022), Fusarium infections in wheat present a global challenge, causing reduced yield, compromised quality, and the accumulation of harmful mycotoxins (Bota et al., 2021; Riaz Ejaz et al., 2023). Fusarium culmorum, a soil-borne fungus (Antalová et al., 2020), is a primary wheat pathogen, with Fusarium root rot, Fusarium crown rot, and Fusarium head blight (FHB) being particularly threatening due to mycotoxin contamination (Bottalico and Perrone, 2002; Scherm et al., 2013; Antalová et al., 2020). Zearalenone (ZEN) and type B trichothecenes, such as deoxynivalenol (DON), acetyl-deoxynivalenol (3-ADON and 15-ADON), and nivalenol (NIV) (Alisaac et al., 2023) pose severe risks to human health, food security, and economy (Ostry et al., 2017). Therefore, managing Fusarium diseases in cereals, especially wheat, is crucial for ensuring food security and safeguarding human health.

Efforts to combat Fusarium infections in wheat have involved substantial investments in genetic resistance, crop rotation, and other practices (Özdemir, 2022). However, complete host resistance remains elusive (Wagacha and Muthomi, 2007), underscoring the ongoing importance of fungicides (Zubrod et al., 2019). Synthetic fungicides, while effective, pose risks to human health and the environment (Castro et al., 2020; Seepe et al., 2021), forcing the exploration of natural alternatives, which are vital for sustainable crop cultivation. Plant extracts are recognized as safe and offer a promising solution (Abdallah et al., 2018), containing a blend of compounds, including phenolic acids, flavonoids, tannins, terpenes, and alkaloids, that can collaborate to inhibit the growth of phytopathogenic fungi (Deresa and Diriba, 2023). Moreover, plant extracts are highly effective against a wide range of pests and diseases, are relatively easy and cheap to produce, and show low toxicity against non-target organisms, including humans (Suteu et al., 2020). Significantly, these compounds not only limit fungal growth by inhibiting ergosterol biosynthesis as an indicator of fungal biomass (Perkowski et al., 2008) but also inhibit mycotoxin biosynthesis and trigger plant defense responses (Acheuk et al., 2022). However, to monitor the in vivo antifungal properties of a plant extract, it is essential to evaluate the phytotoxic effects on the target plant that requires protection, considering that certain studies have indicated their potential adverse effects.

L. album is a medicinal plant distributed in Europe, Asia, and Africa (Chipeva et al., 2013). To understand the mechanisms of action, researchers identified its chemical constituents and found that it has various chemicals, including iridoid glycosides, phenolic acids, flavonoids, alkaloids, triterpenes, and other compounds with diverse biological properties (Damtoft, 1992; Pereira et al., 2012; Pourmirzaee et al., 2019; Sulborska et al., 2020; Uwineza et al., 2021). In recent in vitro research, L. album flower extracts demonstrated promising effects against Fusarium species, emphasizing its versatile applications (Uwineza et al., 2023). Most studies focus on plant extracts' fungicidal or fungistatic effects (García-Ramírez et al., 2023). However, they often overlook the impact of these extracts on mycotoxin production by pathogenic fungi and phytotoxic effects on the crops. Furthermore, most of these studies are conducted in vitro (Kursa et al., 2022; Uwineza et al., 2022), while in vivo experiments are necessary to confirm the biocontrol effects of such plant extracts, as the data available is limited. In addition, for the plant extract to be effective, accurate identification of the pathogen is crucial. Identifying pathogens based on observed symptoms can be challenging, especially at the early stages of disease. Therefore, molecular methods, particularly those based on polymerase chain reaction (PCR), offer superior detection capabilities. The technique is a rapid and sensitive tool for detecting and identifying targeted DNA molecules of pathogens in plant tissues (Pszczółkowska et al., 2013).

The study aimed to assess the efficacy of L. album flower extract [obtained by using supercritical fluid carbon dioxide (SC-CO₂)] on wheat seed germination and analyze its antifungal action against F. culmorum in artificially infected wheat seedlings. Our previous research has shown a promising in vitro effect of L. album against F. culmorum and F. proliferatum (Uwineza et al., 2023), and the literature has indicated that infections in seedlings and basal stems often originate from seed or soil-borne inoculum, leading to significant yield losses due to damaged seedlings, pre-harvest lodging, and impaired grain filling (Gebremariam et al., 2020). Therefore, controlling Fusarium infection from the early stage may promise better harvesting.

The germination capability of wheat seeds serves as a crucial indicator reflecting their quality, viability, and overall potential (Hassani et al., 2019). In our study, L. album decreased seed germination and seedling growth after seven days of incubation compared to the control. This effect was in agreement with other studies, showing reduced wheat germination of seeds treated with Ulva linza or Corallina officinalis seaweeds (Hamouda et al., 2022), Flaveria bidentis (Dai et al., 2022) and aqueous extracts of weed plants: Hyptis sauveolens (L.), Ricinus communis (L.), Alternanthera sessilis (L.), Ipomoea carnea (Jacq), Malachra capitata (L.), and Cymbopogon citrutus (Stapf) (Joshi and Joshi, 2016). The decrease in germination percentage could be attributed to the potential phytotoxic or allelopathic effects of the extracts (Joshi and Joshi, 2016; Ma et al., 2011; Dai et al., 2022; Hamouda et al., 2022; Rys et al., 2022), such as the harmful or inhibitory impact that plant extracts may have on plant tissues or biological processes (Ma et al., 2011; Werrie et al., 2020) or chemical interactions between plants, which may be direct or indirect, beneficial or detrimental (Joshi and Joshi, 2016; Aurelio et al., 2022). The control group exhibited the longest wheat seedlings, with the lowest recorded dry weight compared to the treated wheat seeds. Although plant extracts mildly inhibited seedling weight loss (Figure 1), the effect was not significantly different from the control at p < 0.05. The observed reduction in plant growth under natural conditions may be attributed to seed competition for water, minerals, oxygen, or root system space (Rys et al., 2022). Some plant extracts, despite their beneficial properties, can also contain compounds that hinder seed germination or seedling growth (Bota et al., 2021). Hamouda et al. (2022) investigated the effect of wheat seed priming with the aqueous extracts of Ulva linza and Corallina officinalis seaweeds; higher concentrations inhibited seedling growth and caused specific chromosomal abnormalities, while low concentrations of seaweed extract as a priming treatment improved wheat seedling growth, physiological features and had a greater capacity to boost bioavailable macro- and micronutrients to the plant (Hamouda et al., 2022).

F. culmorum can impact plants across different developmental stages, leading to seedling blight and root rot, hindering seedling emergence and overall plant development (Pastuszak et al., 2021). This pathogenic fungus infects wheat plants, leading to foot rot symptoms, particularly affecting the roots and lower stems. The severity of the disease varies, with early infections causing pre- and post-emergence seedling death, while later infections result in brown lesions on the lower stems and tiller abortion (Scherm et al., 2013). While FHB is often a primary focus in research due to its economic impact and mycotoxin production (Bottalico and Perrone, 2002; Scherm et al., 2013; Antalová et al., 2020), foot rot caused by F. culmorum is a significant issue that requires attention (Scherm et al., 2013). The presence of L. album and F. culmorum infection caused stress to the growing seedlings and by responding to these biotic stresses, seedlings produced many short roots that have influenced the weight and length of the roots (Table 2). Roots are crucial for nutrient uptake and overall plant health. The observed decrease in root length for the seedling treated with 10%_FC may be a defense response triggered by the plant in the presence of Fusarium. L. album extract might influence this response positively, aiding the wheat seedling in resisting fungi infection. Moreover, shorter roots may limit the pathogen's ability to invade the plant's vascular system, acting as a physical barrier. While a decrease in root length might be perceived as an adverse effect of Fusarium infection in wheat, as noted in a study conducted by Saad et al. (2023), it could be a strategic adaptation to strengthen the plants' defense mechanisms induced by plant extract against Fusarium spp, ultimately contributing to enhanced resistance. Previous research showed that seedlings exhibiting moderate vigor were more susceptible to infection than those with high vigor and exhibited a significant height increase when subjected to inoculation with F. graminearum spores (Zhou et al., 2018). Similarly, the application of essential oil disturbed the growth of seedlings' roots and reduced the vigor index (Grzanka et al., 2021). Seedlings protected with L. album extracts showed no symptoms. In contrast, the control samples exhibited visible symptoms, such as root browning, yellowing of the first leaves, and visible mycelia on the above-ground part of the seedlings (Figure 4). Previous studies reported that the early infection reduced wheat seedling growth as F. culmorum can effectively penetrate seedling roots, migrate from hypocotyl to the upper stem internodes and leaves, colonize the host's tissue and cells, block the vascular bundles, disturb nutritional supply and metabolic processes (Kthiri et al., 2021). In another study, three durum wheat genotypes at seedling and full anthesis stage F. culmorum infection significantly decreased chlorophyll a, b, and carotenoid contents in the leaves (Pastuszak et al., 2021). L. album may also cause adverse effects against F. culmorum, inducing toxin accumulation, especially under conditions unfavorable for fungal growth or at low extract concentrations (Tretiakova et al., 2022).

Ergosterol, being integral to fungal cell membranes, serves as an indicator of fungal biomass and is vital for maintaining cell function and integrity (Perkowski et al., 2008; Uwineza et al., 2022). Severe ERG depletion may influence cell growth and proliferation (Abhishek et al., 2015). Here, the effect of L. album extract was evident, as shown by the ERG reduction in infected seedlings. Low ERG levels in non-infected seedlings could be attributed to endophytic fungi—microorganisms living within plant tissues without visible disease symptoms (Abaya et al., 2021; Noel et al., 2022). PCR-based identification confirmed F. oxysporum in the root of infected samples treated with L. album flower extracts. Also, no mycotoxins were identified in those samples. Noel et al. (2022) reported F. oxysporum as one of the fungal endophytes that significantly increased seed weight and lowered the accumulation of DON compared to F. graminearum-infected wheat heads without endophyte (Noel et al., 2022). L. album extract suppressed the growth of pathogenic and endophytic fungi in a concentration-dependent manner. This inhibitory effect could be attributed to the active compounds in L. album flower extracts (Pereira et al., 2012; Pourmirzaee et al., 2019; Sulborska et al., 2020; Uwineza et al., 2021). Detrimental impact on ERG content confirmed a significant disruption or biosynthesis blockage within the plasma membrane of F. culmorum. This finding is in agreement with some of the previous studies that have reported a decrease in ERG content in infected samples after the application of plant extracts (Abhishek et al., 2015; Uwineza et al., 2022). Leaf extract of Solanum torvum Swartz. decreased ERG production by the Fusarium verticillioides strain (Abhishek et al., 2015). Previous in vitro study also showed L. album flower extract activity against F. culmorum and F. proliferatum, reducing ERG content (Uwineza et al., 2023).

Mycotoxigenic fungi can simultaneously produce various compounds (Streit et al., 2012). To monitor whether L. album extract alters the mycotoxin biosynthesis in wheat seedlings, an analysis was conducted, revealing that the content of mycotoxins, including DON, 3- and 15-AcDON, ZEN, and ZEN-14S, commonly associated with F. culmorum contamination (Bryła et al., 2018; Uwineza et al., 2022), were reduced both in roots and leaves of seedlings treated with L. album extract. Abbas and Yli-mattila (2022) showed that the methanolic extract of the medicinal plant Zanthoxylum bungeanum successfully decreased F. graminearum growth and abrogated DON production in wheat heads (Abbas and Yli-mattila, 2022). Similarly, Shcherbakova et al. (2018) found no increase in DON, AcDON, and ZEN content when F. culmorum and F. graminearum developed on extract-treated wheat seeds and seedlings (Shcherbakova et al., 2018).

DON and its acetylated forms 3- and 15-AcDON were completely inhibited by L. album. Correlations between DON and its acetylated forms were highly significant, which is in accordance with the previous research (Sunic et al., 2021), where DON highly correlated with 3-AcDON in winter wheat, and its production by F. culmorum is believed to play a role in pathogenesis (Morimura et al., 2020). Mycotoxin accumulation affects germination rates, seedling growth, pathogen aggressiveness, and overall disease severity (Bruins et al., 1993; Scherm et al., 2011; Winter et al., 2019). Scherm et al. (2011) showed that DON is an aggressiveness factor in F. culmorum stem base infections of durum wheat, whereby disease incidence was decreased by 40%−80% when the trichothecene regulatory gene Tri6 was silenced (Scherm et al., 2011). Similarly, Winter et al. (2019) showed that high DON- and 3-AcDON-producing isolate led to more severe symptoms and 20 times more significant colonization of the stem base than isolates that produced less DON/3-AcDON (Winter et al., 2019). A 10-fold higher accumulation of ZEN and ZEN-14S in infected seedling roots was observed compared to DON, which is consistent with previous findings (Ksieniewicz-Wozniak et al., 2021). It can be explained by L. album extract ability to regulate the enzymes involved in the synthesis or modification of ZEN and its derivatives compared to the ones that process DON. Additionally, the accumulation of ZEN and ZEN-14S over DON may be favored, according to a study that investigated the effect of compactin on mycotoxin production and the expression of associated biosynthetic and regulatory genes in toxigenic F. culmorum. Their findings showed that compactin had a suppressing effect on DON and ZEN differently due to its effect on various genes involved in the biosynthesis of these mycotoxins (Stakheev et al., 2022). These intricate interactions could lead to differential expression of genes related to mycotoxin biosynthesis and modification, resulting in the observed variations in mycotoxin levels (Scherm et al., 2011). However, even though ZEN and ZEN-14S were highly synthesized, the application of L. album significantly reduced them compared to the control. ZEN was reduced to 82.14% for 5% and 87.41% for 10% of L. album extract, and ZEN-14S was decreased to 75.03% for 5% and 78.97% for 10%, confirming the mechanism of action of L. album extract on the reduction of mycotoxin biosynthesis (Jafarzadeh et al., 2022). A strong correlation between ZEN and ZEN-14S was observed, while a weaker correlation occurred among DON, ZEN, and ZEN-14S, which is in accordance with previous research (Birr et al., 2021). ZEN can be modified (e.g., via conjugation) in plants, fungi, and animals through phase I and phase II metabolism (Veršilovskis et al., 2019). This conjugated form (ZEN-14S) is approximately 60 times more estrogenic than ZEN and can be easily hydrolyzed to ZEN in the gastrointestinal tract, thereby increasing exposure to ZEN (Veršilovskis et al., 2019). A high ZEN-14S/ZEN ratio in the malted wheat suggests a possibility of Fusarium converting ZEN into a phase II metabolite through sulfation reactions (Ksieniewicz-Wozniak et al., 2021). Wheat infections occur during or shortly after flowering (Wegulo, 2012); however, our study revealed that mycotoxin accumulation can start at the initial stages of infection.

In this study, we have presented the dual effect of L. album extracts, which acted as antifungal on F. culmorum in wheat seedlings and showed the phytotoxic effect on seed germination. Composition and concentration of L. album extracts can be essential here, as they have not been analyzed until now, but similar results have been reported previously (Bayar and Yilar, 2019; El-Alam et al., 2020; Mehdizadeh et al., 2020). El-Alam et al. (2020) reported that all tested essential oils (EO) presented antifungal properties against F. culmorum by direct contact and volatility assays. However, all tested EO have also shown a phytotoxic activity, either by inhibiting seed germination or affecting root elongation of rye and lettuce (El-Alam et al., 2020). Therefore, carefully considering and optimizing the extract concentration and application method is advised to ensure that the antifungal benefits outweigh any potential phytotoxic effects of the plant extracts.

The present study highlights the dual impact of L. album flower extracts on wheat seed germination, seedling growth, and Fusarium infection control. While exhibiting phytotoxic and allelopathic effects that hindered seed germination and seedling growth, the extracts effectively controlled F. culmorum infection and reduced visible symptoms. The anti-mycotoxigenic potential of L. album flower extracts was evident in the complete inhibition of DON, 3- and 15-AcDON, and the substantial reduction of ZEN and ZEN-14S in artificially infected wheat seedlings. The reduction of ergosterol content in the seedling roots confirmed the disruptive impact on F. culmorum, emphasizing the potential of L. album as a biocontrol agent against Fusarium mycotoxins. The study highlights intricate molecular mechanisms at play and emphasizes the need for further exploration of the effect of L. album extracts on key regulatory genes for mycotoxin biosynthesis. Despite the observed phytotoxicity, the study suggests the potential of L. album extracts as a biocontrol agent, stressing the importance of optimizing concentration and application methods in agricultural practices. Overall, this research contributes valuable insights into the sustainable use of L. album in combating Fusarium species in wheat cultivation, with further exploration required for effective implementation.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

PAU: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. MU: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft. ŁS: Resources, Writing – review & editing. AG-M: Data curation, Validation, Writing – review & editing. AW: Conceptualization, Data curation, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing.